Recombinant microorganisms for the enhanced production of mevalonate, isoprene, isoprenoid precursors, isoprenoids, and acetyl-CoA-derived products

ABSTRACT

The invention features compositions and methods for the increased production of mevalonate, isoprene, isoprenoid precursor molecules, isoprenoids, and/or acetyl-CoA-derived products in recombinant microorganisms by engineering the microorganisms to comprise one or more acetylating proteins such that the expression and/or activity of the one or more acetylating proteins is modulated.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/120,037, filed Jan. 30, 2017, now U.S. Pat. No. 10,648,004, which isa national stage application, filed under 35 U.S.C. § 371, ofInternational Application No. PCT/US2015/016954, filed Feb. 20, 2015,which claims priority to and the benefit of U.S. Provisional ApplicationSer. No. 61/942,546, filed Feb. 20, 2014, the contents of which arehereby fully incorporated by reference.

FIELD OF THE INVENTION

This disclosure relates to compositions and methods for the increasedproduction of mevalonate, isoprene, isoprenoids, isoprenoid precursors,and/or acetyl-CoA-derived products in recombinant microorganisms, aswell as methods for producing and using the same.

INCORPORATION BY REFERENCE TO SEQUENCE LISTING

In the instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 19, 2020, isnamed Substitute_SL_ST25.txt, and is 204,400 bytes in size.

BACKGROUND OF THE INVENTION

Mevalonate is an intermediate of the mevalonate-dependent biosyntheticpathway that converts acetyl-CoA to isopentenyl diphosphate anddimethylallyl diphosphate. The conversion of acetyl-CoA to mevalonatecan be catalyzed by the thiolase, HMG-CoA synthase and the HMG-CoAreductase activities of the upper mevalonate-dependent biosyntheticpathway (MVA pathway).

Commercially, mevalonate has been used as an additive in cosmetics, forthe production of biodegradable polymers, and can have value as a chiralbuilding block for the synthesis of other chemicals.

The products of the mevalonate-dependent pathway are isopentenylpyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP). IPP and DMAPPare precursors to isoprene as well as isoprenoids. Isoprene(2-methyl-1,3-butadiene) is the monomer of natural rubber and also acommon structural motif to an immense variety of other naturallyoccurring compounds, collectively termed the isoprenoids. Isoprene isadditionally the critical starting material for a variety of syntheticpolymers, most notably synthetic rubbers.

Isoprenoids are compounds derived from the isoprenoid precursormolecules IPP and DMAPP. Over 29,000 isoprenoid compounds have beenidentified and new isoprenoids are being discovered each year.Isoprenoids can be isolated from natural products, such asmicroorganisms and species of plants that use isoprenoid precursormolecules as a basic building block to form the relatively complexstructures of isoprenoids. Isoprenoids are vital to most livingorganisms and cells, providing a means to maintain cellular membranefluidity and electron transport. In nature, isoprenoids function inroles as diverse as natural pesticides in plants to contributing to thescents associated with cinnamon, cloves, and ginger. Moreover, thepharmaceutical and chemical communities use isoprenoids aspharmaceuticals, nutraceuticals, flavoring agents, and agricultural pestcontrol agents. Given their importance in biological systems andusefulness in a broad range of applications, isoprenoids have been thefocus of much attention by scientists.

Conventional means for obtaining mevalonate and isoprenoids includeextraction from biological materials (e.g., plants, microbes, andanimals) and partial or total organic synthesis in the laboratory. Suchmeans, however, have generally proven to be unsatisfactory. Inparticular for isoprenoids, given the often times complex nature oftheir molecular structure, organic synthesis is impractical given thatseveral steps are usually required to obtain the desired product.Additionally, these chemical synthesis steps can involve the use oftoxic solvents as can extraction of isoprenoids from biologicalmaterials. Moreover, these extraction and purification methods usuallyresult in a relatively low yield of the desired isoprenoid, asbiological materials typically contain only minute amounts of thesemolecules. Unfortunately, the difficulty involved in obtainingrelatively large amounts of isoprenoids has limited their practical use.

Recent developments in the production of isoprene, isoprenoid precursormolecules, and isoprenoids disclose methods for the production ofisoprene and isoprenoids at rates, titers, and purities that can besufficient to meet the demands of robust commercial processes (see, forexample, International Patent Application Publication No. WO 2009/076676A2 and U.S. Pat. No. 7,915,026); however, improvements to increase theproduction of isoprene and isoprenoids and to increase yields of thesame are still needed.

Such improvements are provided herein by the disclosure of compositionsand methods to increase production of mevalonate as an intermediate ofthe mevalonate-dependent biosynthetic pathway; to increase production ofmolecules derived from mevalonate, such as isoprene, isoprenoidprecursors, and/or isoprenoids; and to increase production ofacetyl-CoA-derived products.

Throughout this specification, various patents, patent applications andother types of publications (e.g., journal articles) are referenced. Thedisclosure of all patents, patent applications, and publications citedherein are hereby incorporated by reference in their entirety for allpurposes.

SUMMARY OF THE INVENTION

The invention provided herein discloses, inter alia, compositions andmethods for the increased production of mevalonate, isoprene, isoprenoidprecursors, isoprenoids, and/or an acetyl-CoA-derived products in amicroorganism by using one or more specific gene manipulations inrecombinant microorganisms/recombinant cells such that the expressionand/or activity of one or more acetylating proteins in the recombinantmicroorganisms is modulated. Such modulation can result in increasedproduction of mevalonate, isoprene, isoprenoid precursor molecules,isoprenoids, and/or acetyl-CoA-derived products.

Accordingly, in one aspect, provided herein are recombinant cellscapable of producing isoprene, wherein the cells comprise: (i) eitherone or more nucleic acids encoding one or more acetylating proteins,wherein the cells have been modified or engineered such that theexpression of the nucleic acids and/or activity of the acetylatingprotein(s) is modulated or one or more acetylating proteins wherein theproteins are engineered such that their activity is modulated; (ii) oneor more nucleic acids encoding one or more polypeptides of the MVApathway; and (iii) a heterologous nucleic acid encoding an isoprenesynthase polypeptide or a polypeptide having isoprene synthase activity,wherein culturing of the recombinant cells in a suitable media providesfor the production of isoprene.

In some embodiments of any of the embodiments disclosed herein, theactivity of the one or more acetylating proteins is modulated such thatthe activity of the one or more acetylating proteins is attenuated,deleted or increased.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is an acetyltransferase. In some embodiments of anyof the embodiments disclosed herein, the acetyltransferase is chosenfrom the group consisting of YfiQ, Pat, and AcuA. In some embodiments ofany of the embodiments disclosed herein, the acetyltransferase is a YfiQpolypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a deacetylase. In some embodiments of any of theembodiments disclosed herein, the deacetylase is chosen from the groupconsisting of CobB and SrtN. In some embodiments of any of theembodiments disclosed herein, the deacetylase is a CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, the oneor more acetylating proteins is selected from the group consisting of aYfiQ polypeptide and a CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a YfiQ polypeptide. In some embodiments of any ofthe embodiments disclosed herein, the activity of the YfiQ polypeptideis modulated by decreasing, attenuating, or deleting the expression ofthe nucleic acid encoding the YfiQ polypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a CobB polypeptide. In some embodiments of any ofthe embodiments disclosed herein, the activity of the CobB polypeptideis modulated by increasing the expression of the nucleic acid encodingthe CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, one ormore polypeptides of the MVA pathway is selected from (a) an enzyme thatcondenses two molecules of acetyl-CoA to form acetoacetyl-CoA; (b) anenzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA(e.g., HMG synthase); (c) an enzyme that converts HMG-CoA to mevalonate;(d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;(e) an enzyme that converts mevalonate 5-phosphate to mevalonate5-pyrophosphate; and (f) an enzyme that converts mevalonate5-pyrophosphate to isopentenyl pyrophosphate.

In some embodiments of any of the embodiments disclosed herein, theheterologous nucleic acid encoding an isoprene synthase polypeptide or apolypeptide having isoprene synthase activity is a plant isoprenesynthase polypeptide. In some embodiments of any of the embodimentsdisclosed herein, the isoprene synthase polypeptide or the polypeptidehaving isoprene synthase activity is a polypeptide from Pueraria orPopulus or a hybrid, Populus alba x Populus tremula. In some embodimentsof any of the embodiments disclosed herein, the isoprene synthasepolypeptide or the polypeptide having isoprene synthase activity is fromthe organism selected from the group consisting of Pueraria montana,Pueraria lobata, Populus tremuloides, Populus alba, Populus nigra, andPopulus trichocarpa.

In some embodiments, the recombinant cells described herein furthercomprise one or more heterologous nucleic acids encoding a polypeptidehaving phosphoketolase activity. In some embodiments of any of theembodiments disclosed herein, the one or more heterologous nucleic acidsencoding a polypeptide having phosphoketolase activity is capable ofsynthesizing glyceraldehyde 3-phosphate and acetyl phosphate (referredto herein interchangeably as acetylphosphate, acetyl-phosphate,acetyl-P, Ac-P) from xylulose 5-phosphate. In some embodiments of any ofthe embodiments disclosed herein, the one or more heterologous nucleicacids encoding a polypeptide having phosphoketolase activity is capableof synthesizing erythrose 4-phosphate and acetyl phosphate from fructose6-phosphate. In some embodiments of any of the embodiments disclosedherein, the recombinant cells further comprise one or more nucleic acidsencoding one or more 1-deoxy-D-xylulose 5-phosphate (DXP) pathwaypolypeptides.

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more pentosephosphate pathway proteins, wherein the cells have been modified suchthat the expression of the nucleic acids encoding the pentose phosphatepathway proteins and/or the activity of the pentose phosphate pathwayproteins is modulated. In some embodiments, the activity of the one ormore pentose phosphate pathway proteins is increased.

In certain embodiments, the activity of the one or more pentosephosphate pathway proteins is increased by increasing the expression ofone or more nucleic acids encoding the pentose phosphate pathwayproteins. In such embodiments, the one or more nucleic acids encodingthe pentose phosphate pathway proteins is selected from the groupconsisting of transketolase (tktA), transaldolase (talB),ribulose-5-phosphate-epimerase (rpe), and ribose-5-phosphate epimerase(rpiA).

In other embodiments, the activity of the one or more pentose phosphatepathway proteins is decreased. In certain embodiments, the activity ofthe one or more pentose phosphate pathway proteins is decreased bydecreasing, attenuating, or deleting the expression of one or morenucleic acids encoding the pentose phosphate pathway proteins. In suchembodiments, the one or more nucleic acids encoding the pentosephosphate pathway proteins comprises phosphofructokinase (pfkA).

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more acetate cyclingproteins, wherein the cells have been modified such that the expressionof the nucleic acids encoding the acetate cycling proteins and/oractivity of the acetate cycling proteins is modulated.

In some embodiments, the activity of the one or more acetate cyclingproteins is increased. In some embodiments, the activity of the one ormore acetate cycling proteins is increased by increasing the expressionof one or more nucleic acids encoding the acetate cycling proteins. Insuch embodiments, the one or more nucleic acids encoding the acetatecycling proteins can be selected from the group consisting ofacetyl-coenzyme A synthetase (acs), acetate kinase (ackA) andphosphotransacetylate (pta).

In some embodiments, the activity of the one or more acetate cyclingproteins is decreased. In some embodiments, the activity of the one ormore acetate cycling proteins is decreased by decreasing, attenuating,or deleting the expression of one or more nucleic acids encoding theacetate cycling proteins. In such embodiments, the one or more nucleicacids encoding the acetate cycling proteins can be selected from thegroup consisting of phosphotransacetylate (pta), acetate kinase (ackA),and acetate transporter/acetate pump (actP).

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more proteinsselected from the group consisting of: sfcA, maeB, pdhR, aceE, aceF,lpdA, glta, acs, pta, ackA, actP, pfkA, rpe, rpiA, tkta, talB, pgl, edd,and eda, and wherein the cells have been modified such that theexpression of the nucleic acids and/or activity of the proteins ismodulated. In some embodiments, the activity of the one or more of theseproteins is increased by increasing the expression of one or morenucleic acids encoding the one or more proteins. In specificembodiments, the one or more nucleic acids encoding the one or moreproteins to be increased is selected from the group consisting of: ackA,pta, sfcA, maeB, aceE, aceF, lpdA, acs, rpe, rpiA, tkta, talB, and pgl.In some embodiments, the activity of the one or more of these proteinsis decreased by decreasing, attenuating, or deleting the expression ofone or more nucleic acids encoding the one or more proteins. In specificembodiments, the one or more nucleic acids encoding one or more proteinsto be decreased is selected from the group consisting of: pdhR, glta,pta, ackA, actP, pfkA, pgl, edd, and eda.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having isoprene synthase activity,the nucleic acid encoding a polypeptide having phosphoketolase activity,the one or more nucleic acids encoding one or more pentose phosphatepathway proteins, or the one or more nucleic acids encoding one or moreacetate cycling proteins, is placed under an inducible promoter or aconstitutive promoter.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having isoprene synthase activity,the nucleic acid encoding a polypeptide having phosphoketolase activity,the one or more nucleic acids encoding one or more pentose phosphatepathway proteins, or the one or more nucleic acids encoding one or moreacetate cycling proteins, is cloned into one or more multicopy plasmids.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having isoprene synthase activity,the nucleic acid encoding a polypeptide having phosphoketolase activity,the one or more nucleic acids encoding one or more pentose phosphatepathway proteins, or the one or more nucleic acids encoding one or moreacetate cycling proteins, is integrated into a chromosome of the cells.

In some embodiments of any of the embodiments disclosed herein, therecombinant cells are gram-positive bacterial cells or gram-negativebacterial cells. In other embodiments of any of the embodimentsdisclosed herein, the recombinant cells are fungal cells, filamentousfungal cells, algal cells or yeast cells. In some embodiments of any ofthe embodiments disclosed herein, the recombinant cells are selectedfrom the group consisting of Bacillus subtilis, Streptomyces lividans,Streptomyces coelicolor, Streptomyces griseus, Escherichia coli, andPantoea citrea. In some embodiments of any of the embodiments disclosedherein, the recombinant cells are selected from the group consisting ofTrichoderma reesei, Aspergillus oryzae, Aspergillus niger, Saccharomycescerevisieae and Yarrowia lipolytica.

In any of the embodiments described herein, the isoprene production isincreased relative to recombinant cells that have not been modified suchthat the expression of the nucleic acids encoding the acetylatingproteins and/or the activity of the acetylating proteins is modulated.

In any of the embodiments described herein, the isoprene production isincreased by at least 5%, wherein the increased production of isoprenecomprises an increase in: (i) titer, (ii) instantaneous yield, (iii)cumulative yield, (iv) ratio of isoprene to carbon dioxide, (v) specificproductivity, or (vi) cell productivity index.

In other aspects, also provided herein are methods for producingisoprene comprising: (a) culturing the recombinant cell of any of theembodiments disclosed herein under conditions suitable for producingisoprene and (b) producing isoprene. In some embodiments, the methodfurther comprises (c) recovering the isoprene.

In another aspect, provided herein are recombinant cells capable ofproducing an isoprenoid precursor, wherein the cells comprise: (i)either one or more nucleic acids encoding one or more acetylatingproteins, wherein the cells have been modified or engineered such thatthe expression of the nucleic acids and/or activity of the acetylatingprotein(s) is modulated or one or more acetylating proteins wherein theproteins are engineered such that their activity is modulated; and (ii)one or more nucleic acids encoding one or more polypeptides of the MVApathway, wherein culturing of the recombinant cells in a suitable mediaprovides for production of the isoprenoid precursor.

In some embodiments of any of the embodiments disclosed herein, theactivity of the one or more acetylating proteins is modulated such thatthe activity of the one or more acetylating proteins is attenuated,deleted or increased.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is an acetyltransferase. In some embodiments of anyof the embodiments disclosed herein, the acetyltransferase is chosenfrom the group consisting of YfiQ, Pat, and AcuA. In some embodiments ofany of the embodiments disclosed herein, the acetyltransferase is a YfiQpolypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a deacetylase. In some embodiments of any of theembodiments disclosed herein, the deacetylase is chosen from the groupconsisting of CobB and SrtN. In some embodiments of any of theembodiments disclosed herein, the deacetylase is a CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, the oneor more acetylating proteins is selected from the group consisting of aYfiQ polypeptide and a CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a YfiQ polypeptide. In some embodiments of any ofthe embodiments disclosed herein, the activity of the YfiQ polypeptideis modulated by decreasing, attenuating, or deleting the expression ofthe nucleic acid encoding the YfiQ polypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a CobB polypeptide. In some embodiments of any ofthe embodiments disclosed herein, the activity of the CobB polypeptideis modulated by increasing the expression of the nucleic acid encodingthe CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, one ormore polypeptides of the MVA pathway is selected from (a) an enzyme thatcondenses two molecules of acetyl-CoA to form acetoacetyl-CoA; (b) anenzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA(e.g., HMG synthase); (c) an enzyme that converts HMG-CoA to mevalonate;(d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;(e) an enzyme that converts mevalonate 5-phosphate to mevalonate5-pyrophosphate; and (f) an enzyme that converts mevalonate5-pyrophosphate to isopentenyl pyrophosphate.

In certain embodiments, the recombinant cells further comprise one ormore heterologous nucleic acids encoding a polypeptide havingphosphoketolase activity. In some embodiments of any of the embodimentsdisclosed herein, the one or more heterologous nucleic acids encoding apolypeptide having phosphoketolase activity is capable of synthesizingglyceraldehyde 3-phosphate and acetyl phosphate from xylulose5-phosphate. In some embodiments of any of the embodiments disclosedherein, the one or more heterologous nucleic acids encoding apolypeptide having phosphoketolase activity is capable of synthesizingerythrose 4-phosphate and acetyl phosphate from fructose 6-phosphate.

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more pentosephosphate pathway proteins, wherein the cells have been modified suchthat the expression of the nucleic acids encoding the pentose phosphatepathway proteins and/or the activity of the pentose phosphate pathwayproteins is modulated. In some embodiments, the activity of the one ormore pentose phosphate pathway proteins is increased.

In certain embodiments, the activity of the one or more pentosephosphate pathway proteins is increased by increasing the expression ofone or more nucleic acids encoding the pentose phosphate pathwayproteins. In such embodiments, the one or more nucleic acids encodingthe pentose phosphate pathway proteins is selected from the groupconsisting of transketolase (tktA), transaldolase (talB),ribulose-5-phosphate-epimerase (rpe), and ribose-5-phosphate epimerase(rpiA).

In other embodiments, the activity of the one or more pentose phosphatepathway proteins is decreased. In certain embodiments, the activity ofthe one or more pentose phosphate pathway proteins is decreased bydecreasing, attenuating, or deleting the expression of one or morenucleic acids encoding the pentose phosphate pathway proteins. In suchembodiments, the one or more nucleic acids encoding the pentosephosphate pathway proteins comprises phosphofructokinase (pfkA).

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more acetate cyclingproteins, wherein the cells have been modified such that the expressionof the nucleic acids encoding the acetate cycling proteins and/oractivity of the acetate cycling proteins is modulated.

In some embodiments, the activity of the one or more acetate cyclingproteins is increased. In some embodiments, the activity of the one ormore acetate cycling proteins is increased by increasing the expressionof one or more nucleic acids encoding the acetate cycling proteins. Insuch embodiments, the one or more nucleic acids encoding the acetatecycling proteins can be selected from the group consisting ofacetyl-coenzyme A synthetase (acs), acetate kinase (ackA) andphosphotransacetylate (pta).

In some embodiments, the activity of the one or more acetate cyclingproteins is decreased. In some embodiments, the activity of the one ormore acetate cycling proteins is decreased by decreasing, attenuating,or deleting the expression of one or more nucleic acids encoding theacetate cycling proteins. In such embodiments, the one or more nucleicacids encoding the acetate cycling proteins can be selected from thegroup consisting of phosphotransacetylate (pta), acetate kinase (ackA),and acetate transporter/acetate pump (actP).

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more proteinsselected from the group consisting of: sfcA, maeB, pdhR, aceE, aceF,lpdA, glta, acs, pta, ackA, actP, pfkA, rpe, rpiA, tkta, talB, pgl, edd,and eda, and wherein the cells have been modified such that theexpression of the nucleic acids and/or activity of the proteins ismodulated. In some embodiments, the activity of the one or more of theseproteins is increased by increasing the expression of one or morenucleic acids encoding the one or more proteins. In specificembodiments, the one or more nucleic acids encoding the one or moreproteins to be increased is selected from the group consisting of: ackA,pta, sfcA, maeB, aceE, aceF, lpdA, acs, rpe, rpiA, tkta, talB, and pgl.In some embodiments, the activity of the one or more of these proteinsis decreased by decreasing, attenuating, or deleting the expression ofone or more nucleic acids encoding the one or more proteins. In specificembodiments, the one or more nucleic acids encoding one or more proteinsto be decreased is selected from the group consisting of: pdhR, glta,pta, ackA, actP, pfkA, pgl, edd, and eda.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having phosphoketolase activity, theone or more nucleic acids encoding one or more pentose phosphate pathwayproteins, or the one or more nucleic acids encoding one or more acetatecycling proteins, is placed under an inducible promoter or aconstitutive promoter.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having phosphoketolase activity, theone or more nucleic acids encoding one or more pentose phosphate pathwayproteins, or the one or more nucleic acids encoding one or more acetatecycling proteins, is cloned into one or more multicopy plasmids.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having phosphoketolase activity, theone or more nucleic acids encoding one or more pentose phosphate pathwayproteins, or the one or more nucleic acids encoding one or more acetatecycling proteins, is integrated into a chromosome of the cells.

In some embodiments of any of the embodiments disclosed herein, therecombinant cells are gram-positive bacterial cells or gram-negativebacterial cells. In other embodiments of any of the embodimentsdisclosed herein, the recombinant cells are fungal cells, filamentousfungal cells, algal cells or yeast cells. In some embodiments of any ofthe embodiments disclosed herein, the recombinant cells are selectedfrom the group consisting of Bacillus subtilis, Streptomyces lividans,Streptomyces coelicolor, Streptomyces griseus, Escherichia coli, andPantoea citrea. In some embodiments of any of the embodiments disclosedherein, the recombinant cells are selected from the group consisting ofTrichoderma reesei, Aspergillus oryzae, Aspergillus niger, Saccharomycescerevisieae and Yarrowia lipolytica.

In some embodiments of any of the embodiments disclosed herein, theisoprenoid precursor is selected from the group consisting of mevalonate(MVA), dimethylallyl diphosphate (DMAPP) and isopentenyl pyrophosphate(IPP).

In any of the embodiments described herein, the isoprenoid precursorproduction is increased relative to recombinant cells that have not beenmodified such that the expression of the nucleic acids encoding theacetylating proteins and/or the activity of the acetylating proteins ismodulated.

In any of the embodiments described herein, the isoprenoid precursorproduction is increased by at least 5%, wherein the increased productionof isoprenoid precursor comprises an increase in: (i) titer, (ii)instantaneous yield, (iii) cumulative yield, (iv) specific productivity,or (v) cell productivity index.

In further aspects, provided herein are methods for producing anisoprenoid precursor comprising: (a) culturing the recombinant cell ofany of the embodiments disclosed herein under conditions suitable forproducing an isoprenoid precursor and (b) producing the isoprenoidprecursor. In some embodiments, the method further comprises (c)recovering the isoprenoid precursor.

In yet another aspect, provided herein are recombinant cells capable ofproducing an isoprenoid, wherein the cells comprise: (i) either one ormore nucleic acids encoding one or more acetylating proteins, whereinthe cells have been modified or engineered such that the expression ofthe nucleic acids and/or activity of the acetylating protein(s) ismodulated or one or more acetylating proteins wherein the proteins areengineered such that their activity is modulated; (ii) one or morenucleic acids encoding one or more polypeptides of the MVA pathway; and(iii) one or more nucleic acids encoding a polyprenyl pyrophosphatesynthase, wherein culturing of the recombinant cells in a suitable mediaprovides for production of the isoprenoid.

In some embodiments of any of the embodiments disclosed herein, theactivity of the one or more acetylating proteins is modulated such thatthe activity of the one or more acetylating proteins is attenuated,deleted or increased.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is an acetyltransferase. In some embodiments of anyof the embodiments disclosed herein, the acetyltransferase is chosenfrom the group consisting of YfiQ, Pat, and AcuA. In some embodiments ofany of the embodiments disclosed herein, the acetyltransferase is a YfiQpolypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a deacetylase. In some embodiments of any of theembodiments disclosed herein, the deacetylase is chosen from the groupconsisting of CobB and SrtN. In some embodiments of any of theembodiments disclosed herein, the deacetylase is a CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, the oneor more acetylating proteins is selected from the group consisting of aYfiQ polypeptide and a CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a YfiQ polypeptide. In some embodiments of any ofthe embodiments disclosed herein, the activity of the YfiQ polypeptideis modulated by decreasing, attenuating, or deleting the expression ofthe nucleic acid encoding the YfiQ polypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a CobB polypeptide. In some embodiments of any ofthe embodiments disclosed herein, the activity of the CobB polypeptideis modulated by increasing the expression of the nucleic acid encodingthe CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, one ormore polypeptides of the MVA pathway is selected from (a) an enzyme thatcondenses two molecules of acetyl-CoA to form acetoacetyl-CoA; (b) anenzyme that condenses acetoacetyl-CoA with acetyl-CoA to form HMG-CoA(e.g., HMG synthase); (c) an enzyme that converts HMG-CoA to mevalonate;(d) an enzyme that phosphorylates mevalonate to mevalonate 5-phosphate;(e) an enzyme that converts mevalonate 5-phosphate to mevalonate5-pyrophosphate; and (f) an enzyme that converts mevalonate5-pyrophosphate to isopentenyl pyrophosphate.

In certain embodiments, the recombinant cells further comprise one ormore heterologous nucleic acids encoding a polypeptide havingphosphoketolase activity. In some embodiments of any of the embodimentsdisclosed herein, the one or more heterologous nucleic acids encoding apolypeptide having phosphoketolase activity is capable of synthesizingglyceraldehyde 3-phosphate and acetyl phosphate from xylulose5-phosphate. In some embodiments of any of the embodiments disclosedherein, the one or more heterologous nucleic acids encoding apolypeptide having phosphoketolase activity is capable of synthesizingerythrose 4-phosphate and acetyl phosphate from fructose 6-phosphate.

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more pentosephosphate pathway proteins, wherein the cells have been modified suchthat the expression of the nucleic acids encoding the pentose phosphatepathway proteins and/or the activity of the pentose phosphate pathwayproteins is modulated. In some embodiments, the activity of the one ormore pentose phosphate pathway proteins is increased.

In certain embodiments, the activity of the one or more pentosephosphate pathway proteins is increased by increasing the expression ofone or more nucleic acids encoding the pentose phosphate pathwayproteins. In such embodiments, the one or more nucleic acids encodingthe pentose phosphate pathway proteins is selected from the groupconsisting of transketolase (tktA), transaldolase (talB),ribulose-5-phosphate-epimerase (rpe), and ribose-5-phosphate epimerase(rpiA).

In other embodiments, the activity of the one or more pentose phosphatepathway proteins is decreased. In certain embodiments, the activity ofthe one or more pentose phosphate pathway proteins is decreased bydecreasing, attenuating, or deleting the expression of one or morenucleic acids encoding the pentose phosphate pathway proteins. In suchembodiments, the one or more nucleic acids encoding the pentosephosphate pathway proteins comprises phosphofructokinase (pfkA).

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more acetate cyclingproteins, wherein the cells have been modified such that the expressionof the nucleic acids encoding the acetate cycling proteins and/oractivity of the acetate cycling proteins is modulated.

In some embodiments, the activity of the one or more acetate cyclingproteins is increased. In some embodiments, the activity of the one ormore acetate cycling proteins is increased by increasing the expressionof one or more nucleic acids encoding the acetate cycling proteins. Insuch embodiments, the one or more nucleic acids encoding the acetatecycling proteins can be selected from the group consisting ofacetyl-coenzyme A synthetase (acs), acetate kinase (ackA) andphosphotransacetylate (pta).

In some embodiments, the activity of the one or more acetate cyclingproteins is decreased. In some embodiments, the activity of the one ormore acetate cycling proteins is decreased by decreasing, attenuating,or deleting the expression of one or more nucleic acids encoding theacetate cycling proteins. In such embodiments, the one or more nucleicacids encoding the acetate cycling proteins can be selected from thegroup consisting of phosphotransacetylate (pta), acetate kinase (ackA),and acetate transporter/acetate pump (actP).

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more proteinsselected from the group consisting of: sfcA, maeB, pdhR, aceE, aceF,lpdA, glta, acs, pta, ackA, actP, pfkA, rpe, rpiA, tkta, talB, pgl, edd,and eda, and wherein the cells have been modified such that theexpression of the nucleic acids and/or activity of the proteins ismodulated. In some embodiments, the activity of the one or more of theseproteins is increased by increasing the expression of one or morenucleic acids encoding the one or more proteins. In specificembodiments, the one or more nucleic acids encoding the one or moreproteins to be increased is selected from the group consisting of: ackA,pta, sfcA, maeB, aceE, aceF, lpdA, acs, rpe, rpiA, tkta, talB, and pgl.In some embodiments, the activity of the one or more of these proteinsis decreased by decreasing, attenuating, or deleting the expression ofone or more nucleic acids encoding the one or more proteins. In specificembodiments, the one or more nucleic acids encoding one or more proteinsto be decreased is selected from the group consisting of: pdhR, glta,pta, ackA, actP, pfkA, pgl, edd, and eda.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, theone or more nucleic acids encoding one or more pentose phosphate pathwayproteins, or the one or more nucleic acids encoding one or more acetatecycling proteins, is placed under an inducible promoter or aconstitutive promoter.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having phosphoketolase activity, theone or more nucleic acids encoding one or more pentose phosphate pathwayproteins, or the one or more nucleic acids encoding one or more acetatecycling proteins, is cloned into one or more multicopy plasmids.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having phosphoketolase activity, theone or more nucleic acids encoding one or more pentose phosphate pathwayproteins, or the one or more nucleic acids encoding one or more acetatecycling proteins, is integrated into a chromosome of the cells.

In some embodiments of any of the embodiments disclosed herein, therecombinant cells are gram-positive bacterial cells or gram-negativebacterial cells. In other embodiments of any of the embodimentsdisclosed herein, the recombinant cells are fungal cells, filamentousfungal cells, algal cells or yeast cells. In some embodiments of any ofthe embodiments disclosed herein, the recombinant cells are selectedfrom the group consisting of Bacillus subtilis, Streptomyces lividans,Streptomyces coelicolor, Streptomyces griseus, Escherichia coli, andPantoea citrea. In some embodiments of any of the embodiments disclosedherein, the recombinant cells are selected from the group consisting ofTrichoderma reesei, Aspergillus oryzae, Aspergillus niger, Saccharomycescerevisieae and Yarrowia lipolytica. In some embodiments of any of theembodiments disclosed herein, the isoprenoid is selected from the groupconsisting of monoterpenoids, sesquiterpenoids, diterpenoids,sesterterpenoids, triterpenoids, and tetraterpenoids. In someembodiments of any of the embodiments disclosed herein, the isoprenoidis selected from the group consisting of abietadiene, amorphadiene,carene, α-famesene, β-famesene, famesol, geraniol, geranylgeraniol,linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene,sabinene, γ-terpinene, terpindene and valencene.

In any of the embodiments described herein, the isoprenoid production isincreased relative to recombinant cells that have not been modified suchthat the expression of the nucleic acids encoding the acetylatingproteins and/or the activity of the acetylating proteins is modulated.

In any of the embodiments described herein, the isoprenoid production isincreased by at least 5%, wherein the increased production of theisoprenoid comprises an increase in: (i) titer, (ii) instantaneousyield, (iii) cumulative yield, (iv) specific productivity, or (v) cellproductivity index.

In further aspects, provided herein are methods for producing anisoprenoid comprising: (a) culturing the recombinant cell of any of theembodiments disclosed herein under conditions suitable for producing anisoprenoid and (b) producing the isoprenoid. In some embodiments, themethod further comprises (c) recovering the isoprenoid.

In yet other aspects, provided herein are recombinant cells capable ofproducing an acetyl-CoA derived product, wherein the cells comprise: (i)either one or more nucleic acids encoding one or more acetylatingproteins, wherein the cells have been modified or engineered such thatthe expression of the nucleic acids and/or activity of the acetylatingprotein(s) is modulated or one or more acetylating proteins wherein theproteins are engineered such that their activity is modulated; and (ii)one or more heterologous nucleic acids encoding a polypeptide havingphosphoketolase activity, wherein the cells produce the acetyl-CoAderived product. In some embodiments, the cells produce increasedamounts of the acetyl-CoA derived product compared to a cell capable ofproducing the acetyl-CoA derived product that does not comprise (i).

In some embodiments, the acetyl-Co-A derived product is selected fromthe group consisting of fatty acids, phenols, prostaglandins, macrolideantibiotics, isoprene, and isoprenoids.

In some embodiments of any of the embodiments disclosed herein, theactivity of the one or more acetylating proteins is modulated such thatthe activity of the one or more acetylating proteins is attenuated,deleted or increased.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is an acetyltransferase. In some embodiments of anyof the embodiments disclosed herein, the acetyltransferase is chosenfrom the group consisting of YfiQ, Pat, and AcuA. In some embodiments ofany of the embodiments disclosed herein, the acetyltransferase is a YfiQpolypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a deacetylase. In some embodiments of any of theembodiments disclosed herein, the deacetylase is chosen from the groupconsisting of CobB and SrtN. In some embodiments of any of theembodiments disclosed herein, the deacetylase is a CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, the oneor more acetylating proteins is selected from the group consisting of aYfiQ polypeptide and a CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a YfiQ polypeptide. In some embodiments of any ofthe embodiments disclosed herein, the activity of the YfiQ polypeptideis modulated by decreasing, attenuating, or deleting the expression ofthe nucleic acid encoding the YfiQ polypeptide.

In some embodiments of any of the embodiments disclosed herein, theacetylating protein is a CobB polypeptide. In some embodiments of any ofthe embodiments disclosed herein, the activity of the CobB polypeptideis modulated by increasing the expression of the nucleic acid encodingthe CobB polypeptide.

In some embodiments of any of the embodiments disclosed herein, the oneor more heterologous nucleic acids encoding a polypeptide havingphosphoketolase activity is capable of synthesizing glyceraldehyde3-phosphate and acetyl phosphate from xylulose 5-phosphate. In someembodiments of any of the embodiments disclosed herein, the one or moreheterologous nucleic acids encoding a polypeptide having phosphoketolaseactivity is capable of synthesizing erythrose 4-phosphate and acetylphosphate from fructose 6-phosphate.

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more pentosephosphate pathway proteins, wherein the cells have been modified suchthat the expression of the nucleic acids encoding the pentose phosphatepathway proteins and/or the activity of the pentose phosphate pathwayproteins is modulated. In some embodiments, the activity of the one ormore pentose phosphate pathway proteins is increased.

In certain embodiments, the activity of the one or more pentosephosphate pathway proteins is increased by increasing the expression ofone or more nucleic acids encoding the pentose phosphate pathwayproteins. In such embodiments, the one or more nucleic acids encodingthe pentose phosphate pathway proteins is selected from the groupconsisting of transketolase (tktA), transaldolase (talB),ribulose-5-phosphate-epimerase (rpe), and ribose-5-phosphate epimerase(rpiA).

In other embodiments, the activity of the one or more pentose phosphatepathway proteins is decreased. In certain embodiments, the activity ofthe one or more pentose phosphate pathway proteins is decreased bydecreasing, attenuating, or deleting the expression of one or morenucleic acids encoding the pentose phosphate pathway proteins. In suchembodiments, the one or more nucleic acids encoding the pentosephosphate pathway proteins comprises phosphofructokinase (pfkA).

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more acetate cyclingproteins, wherein the cells have been modified such that the expressionof the nucleic acids encoding the acetate cycling proteins and/oractivity of the acetate cycling proteins is modulated.

In some embodiments, the activity of the one or more acetate cyclingproteins is increased. In some embodiments, the activity of the one ormore acetate cycling proteins is increased by increasing the expressionof one or more nucleic acids encoding the acetate cycling proteins. Insuch embodiments, the one or more nucleic acids encoding the acetatecycling proteins can be selected from the group consisting ofacetyl-coenzyme A synthetase (acs), acetate kinase (ackA) andphosphotransacetylate (pta).

In some embodiments, the activity of the one or more acetate cyclingproteins is decreased. In some embodiments, the activity of the one ormore acetate cycling proteins is decreased by decreasing, attenuating,or deleting the expression of one or more nucleic acids encoding theacetate cycling proteins. In such embodiments, the one or more nucleicacids encoding the acetate cycling proteins can be selected from thegroup consisting of phosphotransacetylate (pta), acetate kinase (ackA),and acetate transporter/acetate pump (actP).

In some embodiments, the recombinant cells described herein furthercomprise one or more nucleic acids encoding one or more proteinsselected from the group consisting of: sfcA, maeB, pdhR, aceE, aceF,lpdA, glta, acs, pta, ackA, actP, pfkA, rpe, rpiA, tkta, taB, pgl, edd,and eda, and wherein the cells have been modified such that theexpression of the nucleic acids and/or activity of the proteins ismodulated. In some embodiments, the activity of the one or more of theseproteins is increased by increasing the expression of one or morenucleic acids encoding the one or more proteins. In specificembodiments, the one or more nucleic acids encoding the one or moreproteins to be increased is selected from the group consisting of: ackA,pta, sfcA, maeB, aceE, aceF, lpdA, acs, rpe, rpiA, tkta, talB, and pgl.In some embodiments, the activity of the one or more of these proteinsis decreased by decreasing, attenuating, or deleting the expression ofone or more nucleic acids encoding the one or more proteins. In specificembodiments, the one or more nucleic acids encoding one or more proteinsto be decreased is selected from the group consisting of pdhR, glta,pta, ackA, actP, pfkA, pgl, edd, and eda.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having isoprene synthase activity,the nucleic acid encoding a polypeptide having phosphoketolase activity,the one or more nucleic acids encoding one or more pentose phosphatepathway proteins, or the one or more nucleic acids encoding one or moreacetate cycling proteins, is placed under an inducible promoter or aconstitutive promoter.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having isoprene synthase activity,the nucleic acid encoding a polypeptide having phosphoketolase activity,the one or more nucleic acids encoding one or more pentose phosphatepathway proteins, or the one or more nucleic acids encoding one or moreacetate cycling proteins, is cloned into one or more multicopy plasmids.

In any one of the recombinant cells described herein, the one or morenucleic acids encoding one or more acetylating proteins, the one or morenucleic acids encoding one or more polypeptides of the MVA pathway, thenucleic acid encoding a polypeptide having isoprene synthase activity,the nucleic acid encoding a polypeptide having phosphoketolase activity,the one or more nucleic acids encoding one or more pentose phosphatepathway proteins, or the one or more nucleic acids encoding one or moreacetate cycling proteins, is integrated into a chromosome of the cells.

In some embodiments of any of the embodiments disclosed herein, therecombinant cells are gram-positive bacterial cells or gram-negativebacterial cells. In other embodiments of any of the embodimentsdisclosed herein, the recombinant cells are fungal cells, filamentousfungal cells, algal cells or yeast cells. In some embodiments of any ofthe embodiments disclosed herein, the recombinant cells are selectedfrom the group consisting of Bacillus subtilis, Streptomyces lividans,Streptomyces coelicolor, Streptomyces griseus, Escherichia coli, andPantoea citrea. In some embodiments of any of the embodiments disclosedherein, the recombinant cells are selected from the group consisting ofTrichoderma reesei, Aspergillus oryzae, Aspergillus niger, Saccharomycescerevisieae and Yarrowia lipolytica.

In any of the embodiments described herein, the acetyl-CoA derivedproduct production is increased relative to recombinant cells that havenot been modified such that the expression of the nucleic acids encodingthe acetylating proteins and/or the activity of the acetylating proteinsis modulated.

In another aspect, provided herein are methods for producing anacetyl-CoA derived product comprising: (a) culturing the recombinantcells of any one of the embodiments disclosed herein under conditionssuitable for producing the acetyl-CoA derived product and (b) producingthe acetyl-CoA derived product. In some embodiments, the method furthercomprises (c) recovering the acetyl-CoA derived product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts growth (OD600) for control wild type YfiQ cells versuscells carrying a deletion in the YfiQ gene over four hours. FIG. 1Bdepicts overnight growth for the same cells.

FIG. 2 depicts isoprene specific productivity for control wild type YfiQcells versus cells carrying a deletion in the YfiQ gene over four hours.

FIG. 3 depicts broth concentration of acetate measured in each 15-Lfermentation overtime. MCM2732 (YfiQ delete) (closed diamonds); MCS1227(YfiQ wild type) (open squares).

FIG. 4 depicts specific Glucose Uptake Rate measured in each 15-Lfermentation over time. MCM2732 (yfiQ delete) (closed diamonds); MCS1227(yfiQ wild type) (open squares).

FIG. 5 depicts volumetric isoprene productivity achieved in each 15-Lfermentation over time. MCM2732 (yfiQ delete) (closed diamonds); MCS1227(yfiQ wild type) (open squares).

FIG. 6 depicts cumulative yield of isoprene on glucose achieved in each15-L fermentation over time. MCM2732 (yfiQ delete) (closed diamonds);MCS1227 (yfiQ wild type) (open squares).

FIG. 7 depicts Cell Performance Index (CPI) achieved in each 15-Lfermentation over time. MCM2732 (yfiQ delete) (closed diamonds); MCS1227(yfiQ wild type) (open squares).

FIG. 8 depicts smoothed specific isoprene productivity achieved in each15-L fermentation overtime. MCM2732 (yfiQ delete) (closed diamonds);MCS1227 (yfiQ wild type) (open squares).

FIG. 9 depicts growth rates of yfiQ wild type and yfiQ deletionisoprene-producing cells grown on various concentrations of acetate as asole carbon source.

FIG. 10 depicts specific isoprene productivity of yfiQ wild type andyfiQ deletion isoprene producing cells grown on various concentrationsof acetate as a sole carbon source.

FIG. 11 depicts growth rates of yfiQ wild type and yfiQ deletionisoprene-producing cells grown on glucose as a sole carbon source.

FIG. 12 depicts specific isoprene productivity of yfiQ wild type andyfiQ deletion isoprene-producing cells grown on glucose as a sole carbonsource.

FIG. 13 depicts a map of plasmid pMCS826 PKL16 (M. hommis).

FIG. 14 depicts a map of plasmid pEWL1421.

FIG. 15 depicts a map of plasmid pMCS1019.

FIG. 16 depicts broth concentration of acetate measured in each 15-Lfermentation over time. All cells have yfiQ deleted. MCM2732 controlcells (M. hominis phosphoketolase heterologously express and ptaoverexpress) (closed diamond); MCM3151 (M. hominis phosphoketolaseheterologously express and pta delete) (open squares); MD1206 (M.hominis phosphoketolase heterologously express and pta delete) (opentriangles); MD1207 (E. gallinarum phosphoketolase heterologously expressand pta delete) (lines marked with an ‘x’).

FIG. 17 depicts the cumulative yield of isoprene on glucose achieved ineach 15-L fermentation over time. All cells have yfiQ deleted. MCM2732control cells (M. hominis phosphoketolase heterologously express and ptaoverexpress) (closed diamond); MCM3151 (M. hominis phosphoketolaseheterologously express and pta delete) (open squares); MD1206 (M.hominis phosphoketolase heterologously express and pta delete) (opentriangles); MD1207 (E. gallinarum phosphoketolase heterologously expressand pta delete) (lines marked with an ‘x’)

FIG. 18 depicts isoprene yield on glucose (over previous 40 hr period)achieved in each 15-L fermentation over time. All cells have yfiQdeleted. MCM2732 control cells (M. hominis phosphoketolaseheterologously express and pta overexpress) (closed diamond); MCM3151(M. hominis phosphoketolase heterologously express and pta delete) (opensquares); MD1206 (M. hominis phosphoketolase heterologously express andpta delete) (open triangles); MD1207 (E. gallinarum phosphoketolaseheterologously express and pta delete) (lines marked with an ‘x’)

FIG. 19 depicts maps of the actP deletion and ackA overexpressionallelic exchanges vectors.

FIG. 20A-D depict the growth rate (FIG. 20A), carbon dioxide evolutionrates (CER) over time (FIG. 20B), broth acetate over time (FIG. 20C),and broth MVA over time (FIG. 20D) of MD1207 (control), MD1245 (actPdelete), and DW1245 (ackA overexpress). All cells are yfiQ and ptadeleted with E. gallinarum phosphoketolase heterologous expression.

FIG. 21A-D depict the instantaneous isoprene/CO₂ production (FIG. 21A),isoprene titer produced over time (FIG. 21B), % yield of isopreneproduced over time (FIG. 21C), and isoprene specific productivity overtime (FIG. 21D) of MD1207 (control), MD1245 (actP delete), and DW1245(ackA overexpress). All cells are yfiQ and pta deleted with E.gallinarum phosphoketolase heterologous expression.

FIG. 22 depicts a map of the pentose phosphate pathway upregulationallelic exchange vector.

FIG. 23 depicts a map of the pfkA downregulation allelic exchangevector.

FIG. 24 depicts the growth rate of cells expressing PfkA::TmRNAproteolytic tags with isoleucine (I), arginine (R), or threonine (T)mutations in the third to last amino acid position.

FIG. 25A-D depict the growth rate (FIG. 25A), carbon dioxide evolutionrates (CER) over time (FIG. 25B), broth acetate over time (FIG. 25C),and broth MVA over time (FIG. 25D) of MD1207 (control) and MD1284 (PPPoverexpression). All cells are yfiQ and pta deleted with E. gallinarumphosphoketolase heterologous expression.

FIG. 26A-D depicts the instantaneous isoprene/CO₂ production (FIG. 26A),isoprene titer produced over time (FIG. 26B), % yield of isopreneproduced over time (FIG. 26C), and isoprene specific productivity overtime (FIG. 26D) of MD1207 (control) and MD1284 (PPP overexpression). Allcells are yfiQ and pta deleted with E. gallinarum phosphoketolaseheterologous expression.

FIG. 27A-B depicts the growth rate (FIG. 27A) and isoprene titerproduced over time (FIG. 27B) of MD1207 (control), MD1284 (PPPoverexpression), MD1286 (PPP overexpression and PfkA I tag), MD1285 (PPPoverexpression and PfkA T tag), MD1287 ((PPP overexpression and PfkA Rtag)). All cells are yfiQ and pta deleted with E. gallinarumphosphoketolase heterologous expression.

FIG. 28 depicts metabolism in a cell producing mevalonate, isoprene,isoprenoids, isoprenoid precursors, and/or acetyl-CoA-derived products.

DETAILED DESCRIPTION

The invention provides, inter alia, compositions and methods for theincreased production of mevalonate, isoprene, isoprenoid precursormolecules, isoprenoids, and/or acetyl-CoA-derived products inrecombinant microorganisms that have been engineered for modulatedexpression of genes encoding polypeptides involved in proteinacetylation (“acetylating proteins” or “acetylation proteins”). Morespecifically, the invention provides, inter alia, recombinantmicroorganisms, or progeny thereof, comprising cells wherein theactivity of one or more acetylating proteins is modulated. Theseacetylating proteins can include, without limitation, acetyl transferasepolypeptides (acetyltransferases) and/or deacetylase polypeptides. Insome embodiments, the activity of said one or more acetylating proteinsis modulated such that the activity of said one or more acetylatingproteins is decreased, attenuated, deleted or increased. In someembodiments of any of the embodiments disclosed herein, the one or moreacetylating proteins is selected from the group consisting of a YfiQpolypeptide, a Pat polypeptide, an AcuA polypeptide, a Salmonellaenterica acetyltransferase (gi|16503810|emb|CAD05835.1|SEQ ID NO:51), aRhodopseudomonas palustris GCN5 family N-acetyltransferase(gi|499473135|ref|WP_011159775.1| SEQ ID NO:52), a Streptomyces lividansprotein acetyl transferase (EFD66247 SEQ ID NO:53), a Mycobacteriumtuberculosis acetyltransferase (gi|15608138|ref|NP_215513.1| SEQ IDNO:54), and a Mycobacterium smegmatis acetyl transferase(gi|118468187|ref|YP_889697.1| SEQ ID NO:55). In some embodiments of anyof the embodiments disclosed herein, the one or more acetylatingproteins is selected from the group consisting of a CobB polypeptide, aSrtN polypeptide, a Salmonella enterica NAD-dependent deacetylase(gi|16764576|ref|NP_460191.1| SEQ ID NO:56), a Rhodopseudomonaspalustris NAD-dependent deacetylase (gi|499471434|ref|WP_011158074.11SEQ ID NO:57), and a Mycobacterium tuberculosis NAD-dependent proteindeacylase (gi|614103494|sp|P9WGG3.1|NPD_MYCTU SEQ ID NO:58). In someembodiments of any of the embodiments disclosed herein, the one or moreacetylating proteins is selected from the group consisting of a YfiQpolypeptide and a CobB polypeptide. In some embodiments of any of theembodiments disclosed herein, the acetylating protein is a YfiQpolypeptide. In some embodiments of any of the embodiments disclosedherein, the activity of the YfiQ polypeptide is modulated by decreasing,attenuating, or deleting the expression of the gene/nucleic acidencoding the YfiQ polypeptide. In some embodiments of any of theembodiments disclosed herein, the activity of the YfiQ polypeptide ismodulated by increasing the expression of the gene encoding the YfiQpolypeptide. In some embodiments of any of the embodiments disclosedherein, the acetylating protein is a CobB polypeptide. In someembodiments of any of the embodiments disclosed herein, the activity ofthe CobB polypeptide is modulated by decreasing, attenuating, ordeleting the expression of the gene encoding the CobB polypeptide. Insome embodiments of any of the embodiments disclosed herein, theactivity of the CobB polypeptide is modulated by increasing theexpression of the gene encoding the CobB polypeptide.

As detailed herein, acetylation is a post-translational modificationused by cells to control the activity of proteins as well as to regulategene expression in response to rapidly changing conditions (Cerezo etal., 2011, Molec. Microb., 82(5):1110-28). Acetylation of cellularproteins is controlled by enzymes known as acetyltransferases, whichtransfer the acetyl group of intracellularly available acetyl-CoA onto atarget protein, and deacetylases, which remove acetyl groups from aminoacids in proteins. As disclosed herein, the inventors have discovered,inter alia, that modulation of genes responsible for regulatingintracellular acetylation results in substantial and surprisingimprovements in the production of molecules derived from mevalonate,including isoprene, isoprenoid precursors, and isoprenoids, as well asthe production of molecules derived from acetyl-CoA.

In one aspect, the recombinant microorganisms disclosed herein are cells(such as bacterial, fungal, or algal cells) that have been furtherengineered or modified to heterologously express nucleic acids encodingone or more mevalonate (MVA) pathway polypeptides. Themevalonate-dependent biosynthetic pathway is particularly important forthe production of the isoprenoid precursor molecules dimethylallyldiphosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The enzymes ofthe upper mevalonate pathway convert acetyl-CoA into mevalonate viathree enzymatic reactions. Without being bound to theory, it is believedthat increasing the amount of acetyl-CoA intracellularly available forentrance into the upper mevalonate-dependent biosynthetic pathway willsubstantially increase intracellular concentrations of mevalonate and,consequently, of downstream isoprenoid precursor molecules such as DMAPPand IPP. The increased yield of mevalonate production by these strainsis therefore advantageous for commercial applications. Any progeny ofthe recombinant microorganism is contemplated to be within the scope ofthe invention as well.

Furthermore, modulation of additional genes involved in the utilizationof carbon during cellular metabolism or that are implicated with respectto the available intracellular supply of acetyl-CoA may also bemodulated to improve production of mevalonate, isoprene, isoprenoidprecursors, and/or isoprenoids. These include, but are not limited to,genes encoding phosphoketolase, citrate synthase, phosphotransacetylase,acetate kinase, lactate dehydrogenase, malic enzyme and/or pyruvatedehydrogenase which can be modulated to increase or decrease theactivity of enzymes in metabolic pathways such that more carbon flux isdirected toward mevalonate production. Other factors, the modulation ofwhich can increase carbon flux towards mevalonate in cells, can include6-phosphogluconolactonase, phosphoenolpyruvate carboxylase, theinhibitor of RssB activity during magnesium starvation protein, the AcrAcomponent of the multidrug efflux pump AcrAB-TolC, and the fumarate andnitrate reduction sRNA. This, in turn, can lead to more substrate forthe production of isoprene, isoprenoid precursors, and isoprenoids. Thecompositions and methods of the present application, therefore,represent an improvement over what has previously been practiced in theart, both in the number of strains of microorganisms available forincreased production of mevalonate, isoprene, isoprenoid precursormolecules, isoprenoids, and acetyl-coA-derived products as well as inthe amount of these compounds (e.g., mevalonate) produced by those cells(such as bacterial, fungal, or algal cells).

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, “Molecular Cloning: A LaboratoryManual”, second edition (Sambrook et al., 1989); “OligonucleotideSynthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I.Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.);“Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds.,1987, and periodic updates); “PCR: The Polymerase Chain Reaction”,(Mullis et al., eds., 1994). Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanismsand Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provideone skilled in the art with a general guide to many of the terms used inthe present application.

Definitions

The terms “complete mevalonate (MVA) pathway” or “entire mevalonate(MVA) pathway” refer to the cellular metabolic pathway which convertsacetyl-CoA into dimethylallyl pyrophosphate (DMAPP) and isopentenylpyrophosphate (IPP) and which is catalyzed by the enzymesacetoacetyl-Coenzyme A synthase (e.g., thiolase),3-hydroxy-3-methylglutaryl-Coenzyme A synthase,3-hydroxy-3-methylglutaryl-Coenzyme A reductase, mevalonate kinase(MVK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase(MVD), and isopentenyl diphosphate isomerase (IDI).

As used herein, the terms “upper mevalonate pathway” or “upper MVApathway” refer to the series of reactions in cells catalyzed by theenzymes acetoacetyl-Coenzyme A synthase (e.g., thiolase),3-hydroxy-3-methylglutaryl-Coenzyme A synthase, and3-hydroxy-3-methylglutaryl-Coenzyme A reductase.

The terms “lower mevalonate pathway” or “lower MVA pathway” refer to theseries of reactions in cells catalyzed by the enzymes mevalonate kinase(MVK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase(MVD), and isopentenyl diphosphate isomerase (IDI).

The term “isoprene” refers to 2-methyl-1,3-butadiene (CAS #78-79-5). Itcan be the direct and final volatile C5 hydrocarbon product from theelimination of pyrophosphate from 3,3-dimethylallyl diphosphate (DMAPP).It may not involve the linking or polymerization of IPP molecules toDMAPP molecules. The term “isoprene” is not generally intended to belimited to its method of production unless indicated otherwise herein.

As used herein, the term “polypeptides” includes polypeptides, proteins,peptides, fragments of polypeptides, and fusion polypeptides.

As used herein, an “isolated polypeptide” is not part of a library ofpolypeptides, such as a library of 2, 5, 10, 20, 50 or more differentpolypeptides and is separated from at least one component with which itoccurs in nature. An isolated polypeptide can be obtained, for example,by expression of a recombinant nucleic acid encoding the polypeptide.

By “heterologous polypeptide” is meant a polypeptide encoded by anucleic acid sequence derived from a different organism, species, orstrain than the host cell. In some embodiments, a heterologouspolypeptide is not identical to a wild-type polypeptide that is found inthe same host cell in nature.

As used herein, a “nucleic acid” refers to two or moredeoxyribonucleotides and/or ribonucleotides covalently joined togetherin either single or double-stranded form.

By “recombinant nucleic acid” is meant a nucleic acid of interest thatis free of one or more nucleic acids (e.g., genes) which, in the genomeoccurring in nature of the organism from which the nucleic acid ofinterest is derived, flank the nucleic acid of interest. The termtherefore includes, for example, a recombinant DNA which is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA, a genomic DNA fragment, or a cDNAfragment produced by PCR or restriction endonuclease digestion)independent of other sequences.

By “heterologous nucleic acid” is meant a nucleic acid sequence derivedfrom a different organism, species or strain than the host cell. In someembodiments, the heterologous nucleic acid is not identical to awild-type nucleic acid that is found in the same host cell in nature.For example, a nucleic acid encoded by the mvaE and mvaS genestransformed in or integrated into the chromosome of E. coli is aheterologous nucleic acid.

As used herein, an “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid of interest. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. An expression control sequencecan be “native” or heterologous. A native expression control sequence isderived from the same organism, species, or strain as the gene beingexpressed. A heterologous expression control sequence is derived from adifferent organism, species, or strain as the gene being expressed. An“inducible promoter” is a promoter that is active under environmental ordevelopmental regulation.

By “operably linked” is meant a functional linkage between a nucleicacid expression control sequence (such as a promoter) and a secondnucleic acid sequence, wherein the expression control sequence directstranscription of the nucleic acid corresponding to the second sequence.

As used herein, the terms “minimal medium” or “minimal media” refer togrowth medium containing the minimum nutrients possible for cell growth,generally without the presence of amino acids. Minimal medium typicallycontains: (1) a carbon source for microorganism (e.g., such asbacterial, fungal, or algal cells) growth; (2) various salts, which canvary among microorganism (e.g., bacterial) species and growingconditions; and (3) water. The carbon source can vary significantly,from simple sugars like glucose to more complex hydrolysates of otherbiomass, such as yeast extract, as discussed in more detail below. Thesalts generally provide essential elements such as magnesium, nitrogen,phosphorus, and sulfur to allow the cells to synthesize proteins andnucleic acids. Minimal medium can also be supplemented with selectiveagents, such as antibiotics, to select for the maintenance of certainplasmids and the like. For example, if a microorganism is resistant to acertain antibiotic, such as ampicillin or tetracycline, then thatantibiotic can be added to the medium in order to prevent cells lackingthe resistance from growing. Medium can be supplemented with othercompounds as necessary to select for desired physiological orbiochemical characteristics, such as particular amino acids and thelike.

As used herein, the term “isoprenoid” refers to a large and diverseclass of naturally-occurring class of organic compounds composed of twoor more units of hydrocarbons, with each unit consisting of five carbonatoms arranged in a specific pattern. As used herein, “isoprene” isexpressly excluded from the definition of “isoprenoid.”

As used herein, the term “terpenoid” refers to a large and diverse classof organic molecules derived from five-carbon isoprenoid units assembledand modified in a variety of ways and classified in groups based on thenumber of isoprenoid units used in group members. Hemiterpenoids haveone isoprenoid unit. Monoterpenoids have two isoprenoid units.Sesquiterpenoids have three isoprenoid units. Diterpenoids have fourisoprene units. Sesterterpenoids have five isoprenoid units.Triterpenoids have six isoprenoid units. Tetraterpenoids have eightisoprenoid units. Polyterpenoids have more than eight isoprenoid units.

As used herein, “isoprenoid precursor” refers to any molecule that isused by organisms in the biosynthesis of terpenoids or isoprenoids.Non-limiting examples of isoprenoid precursor molecules include, e.g.,isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP).

As used herein, the term “mass yield” refers to the mass of the productproduced by the cells (such as bacterial, fungal, or algal cells)divided by the mass of the glucose consumed by the cells (such asbacterial, fungal, or algal cells) multiplied by 100.

By “specific productivity,” it is meant the mass of the product producedby the cells (such as bacterial, fungal, or algal cells) divided by theproduct of the time for production, the cell density, and the volume ofthe culture.

By “titer,” it is meant the mass of the product produced by the cells(such as bacterial, fungal, or algal cells) divided by the volume of theculture.

As used herein, the term “cell productivity index (CPI)” refers to themass of the product produced by the cells (such as bacterial, fungal, oralgal cells) divided by the mass of the cells (such as bacterial,fungal, or algal cells) produced in the culture.

As used herein, the term “acetyl-CoA-derived products” refer tosecondary metabolites derived from acetyl-CoA. Examples of secondarymetabolites include, but are not limited to, fatty acids, phenols,prostaglandins, macrolide antibiotics, isoprene, and isoprenoids.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains.

As used herein, the singular terms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

Modulation of Cellular Acetylation Machinery

Acetylation of residues in proteins is a post-translational modificationseen throughout prokaryotic and eukaryotic cells. Acetylation of lysineresidue is a common post-translational modification that is observed. Asa post-translational modification, acetylation can be used by cells tocontrol the activity of proteins as well as to regulate gene expressionin response to rapidly changing conditions (Cerezo et al., 2011, Molec.Microb., 82(5):1110-28). Acetylation of cellular proteins is controlledby enzymes known as acetyltransferases (such as, acetyl-CoA-dependentacetyl transferases or Gcn5-like protein N-acetyltransferases, e.g.,YfiQ) which transfer the acetyl group of a molecule of acetyl-CoA onto atarget protein. As used herein, the term “YfiQ” refers to anacetyltransferase polypeptide encoded by the yfiQ which can be usedinterchangeably with the terms “protein lysine acetylase” or “pka.” Incontrast, deacetylation of intracellular proteins is controlled byenzymes known as deacetylases, such as NAD⁺-dependent (Sir2-like)protein deacetylases (otherwise known as sirtuins, for example, CobB).

One important target for protein acetylation in microorganisms isAMP-forming acetyl-coenzyme A synthetase (Acs), which is a ubiquitousenzyme responsible for the conversion of acetate to the high energyintermediate acetyl-CoA, a keystone molecule of central metabolism(Cerezo et al., 2011, Molec. Microb., 82(5):1110-28). The deacetylaseactivity of CobB has been demonstrated on Acs in vitro (Zhao et al.,2004, J Mol. Biol., 337:731-41). Without being bound to theory, sinceacetylation of Acs results in its enzymatic inactivation, cellsengineered to decrease the amount of Acs acetylation could be expectedto produce higher amounts of acetyl-CoA.

By manipulating the pathways that involves intracellular proteinacetylation, the recombinant microorganism can produce decreased amountsof acetate in comparison to microorganisms that do not have modulatedendogenous acetyltransferase and/or deacetylase gene expression orprotein activity. Decreases in the amount of acetate produced can bemeasured by routine assays known to one of skill in the art. The amountof acetate reduction is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% as compared to cellscomprising one or more nucleic acids encoding one or more acetylatingproteins, wherein said cells have been engineered such that theexpression of the nucleic acids and/or activity of the acetylatingprotein(s) is modulated.

The activity of acetyltransferases (such as, but not limited to YfiQ(pka), Pat, or AcuA) or deacetylases (such as, but not limited to CobBand SrtN) can also be decreased by molecular manipulation of proteinactivity or gene expression. The decrease in protein activity or geneexpression can be any amount of reduction of gene expression, specificactivity or total activity as compared to when no manipulation has beeneffectuated. In some instances, the decrease of enzyme activity isdecreased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

The activity of acetyltransferases (such as, but not limited to YfiQ(pka), Pat, or AcuA) or deacetylases (such as, but not limited to CobBand SrtN) can also be increased by molecular manipulation of proteinactivity or gene expression. The increase in protein activity or geneexpression can be any amount of increase in gene expression, specificactivity or total activity as compared to when no manipulation has beeneffectuated. In some instances, the increase of enzyme activity isincreased by at least about 1%, 5%0, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%,750%, 1000% or more.

In some embodiments, the activity of said one or more acetylatingproteins is modulated such that the activity of said one or moreacetylating proteins is attenuated, deleted or increased. In someembodiments of any of the embodiments disclosed herein, the one or moreacetylating proteins is selected from the group consisting of a YfiQpolypeptide and a CobB polypeptide. In some embodiments of any of theembodiments disclosed herein, the acetylating protein is a YfiQpolypeptide. In some embodiments of any of the embodiments disclosedherein, the activity of the YfiQ polypeptide is modulated by decreasing,attenuating, or deleting the expression of the gene encoding the YfiQpolypeptide. In some embodiments of any of the embodiments disclosedherein, the activity of the YfiQ polypeptide is modulated by increasingthe expression of the gene encoding the YfiQ polypeptide. In someembodiments of any of the embodiments disclosed herein, the acetylatingprotein is a CobB polypeptide. In some embodiments of any of theembodiments disclosed herein, the activity of the CobB polypeptide ismodulated by decreasing, attenuating, or deleting the expression of thegene encoding the CobB polypeptide. In some embodiments of any of theembodiments disclosed herein, the activity of the CobB polypeptide ismodulated by increasing the expression of the gene encoding the CobBpolypeptide.

In some cases, modulating the activity of an acetyltransferase and/or adeacetylase gene (either at the transcriptional (i.e., gene expression)and/or translational level (i.e., protein activity) results in morecarbon flux into the mevalonate dependent biosynthetic pathway incomparison to cells that do not have modulated acetyltransferase and/ora deacetylase activity.

Recombinant Cells Capable of Production of Mevalonate

The mevalonate-dependent biosynthetic pathway (MVA pathway) is a keymetabolic pathway present in all higher eukaryotes and certain bacteria.In addition to being important for the production of molecules used inprocesses as diverse as protein prenylation, cell membrane maintenance,protein anchoring, and N-glycosylation, the mevalonate pathway providesa major source of the isoprenoid precursor molecules DMAPP and IPP,which serve as the basis for the biosynthesis of terpenes, terpenoids,isoprenoids, and isoprene.

In the upper portion of the MVA pathway, acetyl-CoA produced duringcellular metabolism is converted to mevalonate via the actions ofpolypeptides having thiolase, HMG-CoA reductase, and HMG-CoA synthaseenzymatic activity. First, acetyl-CoA is converted to acetoacetyl-CoAvia the action of a thiolase. Next, acetoacetyl-CoA is converted to3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by the enzymatic action ofHMG-CoA synthase. This Co-A derivative is reduced to mevalonate byHMG-CoA reductase, which is the rate-limiting step of the mevalonatepathway of isoprenoid production. Mevalonate is then converted intomevalonate-5-phosphate via the action of mevalonate kinase which issubsequently transformed into mevalonate-5-pyrophosphate by theenzymatic activity of phosphomevalonate kinase. Finally, IPP is formedfrom mevalonate-5-pyrophosphate by the activity of the enzymemevalonate-5-pyrophosphate decarboxylase.

In some aspects, modulation of the any of the enzymes referred to hereincan affect the expression (e.g., transcription or translation),production, post-translational modification or any other function of theenzyme. In some embodiments, the function of the enzyme (e.g., catalyticability) in recombinant cells is increased or decreased as compared to acell that has not been engineered for such modulation. In oneembodiment, the function of the enzyme (e.g. activity) is increased ascompared to a cell that has not been engineered. In another embodiment,the function of the enzyme (e.g. activity) is decreased as compared to acell that has not been engineered.

Any of the enzymes from the upper and lower MVA pathway may be used incombination with the engineered host cells described herein.Non-limiting examples of MVA pathway polypeptides include acetyl-CoAacetyltransferase (AA-CoA thiolase) polypeptides, acetoacetyl-CoAsynthase (nphT7), 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoAsynthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase(HMG-CoA reductase) polypeptides, mevalonate kinase (MVK) polypeptides,phosphomevalonate kinase (PMK) polypeptides, diphosphomevalontedecarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC)polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDIpolypeptides, and polypeptides (e.g., fusion polypeptides) having anactivity of two or more MVA pathway polypeptides. MVA pathwaypolypeptides can include polypeptides, fragments of polypeptides,peptides, and fusions polypeptides that have at least one activity of anMVA pathway polypeptide. Exemplary MVA pathway nucleic acids includenucleic acids that encode a polypeptide, fragment of a polypeptide,peptide, or fusion polypeptide that has at least one activity of an MVApathway polypeptide. Exemplary MVA pathway polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein.

Non-limiting examples of MVA pathway polypeptides which can be used aredescribed in International Patent Application Publication No.WO2009/076676; WO2010/003007 and WO2010/148150.

Genes Encoding MvaE and MvaS Polypeptides

In some microorganisms (such as, but not limited to, L. grayi, E.faecium, E. gallinarum, E. casseliflavus, and E. faecalis), the mvaEgene encodes a polypeptide that possesses both thiolase and HMG-CoAreductase activities. In fact, the mvaE gene product represented thefirst bifunctional enzyme of IPP biosynthesis found in eubacteria andthe first example of HMG-CoA reductase fused to another protein innature (Hedl, et al., J Bacteriol. 2002 April; 184(8): 2116-2122). ThemvaS gene, on the other hand, encodes a polypeptide having an HMG-CoAsynthase activity. The mvaE and mvaS genes of a different bacterialspecies, E. faecalis, have been incorporated into E. coli strainspreviously to produce mevalonate (see US 2005/0287655 A1, the disclosureof which is incorporated by reference herein; Tabata, K. and Hashimoto,S.-I. Biotechnology Letters 26: 1487-1491, 2004).

Accordingly, cells (such as bacterial cells, e.g., E. coli) can beengineered to express one or more mvaE and mvaS genes (such as, but notlimited to, mvaE and mvaS genes from L. grayi, E. faecium, E.gallinarum, E. casseliflavus, and/or E. faecalis), to increaseproduction, peak titer, and cell productivity of mevalonate. The one ormore mvaE and mvaS genes can be expressed on a multicopy plasmid. Theplasmid can be a high copy plasmid, a low copy plasmid, or a medium copyplasmid. Alternatively, the one or more mvaE and mvaS genes can beintegrated into the host cell's chromosome. For both heterologousexpression of the one or more mvaE and mvaS genes on a plasmid or as anintegrated part of the host cell's chromosome, expression of the genescan be driven by either an inducible promoter or a constitutivelyexpressing promoter. The promoter can be a strong driver of expression,it can be a weak driver of expression, or it can be a medium driver ofexpression of the one or more mvaE and mvaS genes.

Any genes encoding an upper MVA pathway polypeptide can be used in thepresent invention. In certain embodiments, various options of mvaE andmvaS genes (such as, but not limited to, mvaE and mvaS genes from L.grayi, E. faecium, E. gallinarum, E. casseliflavus, and/or E. faecalis)alone or in combination with one or more other mvaE and mvaS genesencoding proteins from the upper MVA pathway are contemplated within thescope of the invention. Thus, in certain aspects, any of thecombinations of genes contemplated in Table 1 can be expressed in cells(such as bacterial, fungal, or algal cells) in any of the ways describedabove.

TABLE 1 Options For Expression of MvaE And MvaS Genes In Host CellsContemplated L. grayi, E. faecium, E. gallinarum, E. casseliflavus, E.faecalis, mvaE mvaE mvaE mvaE mvaE L. grayi, L. grayi, E. faecium, E.gallinarum, E. casseliflavus, E. faecalis, mvaS mvaE mvaE mvaE mvaE mvaEL. grayi, L. grayi, L. grayi, L. grayi, L. grayi, mvaS mvaS mvaS mvaSmvaS E. faecium, L. grayi, E. faecium, E. gallinarum, E. casseliflavus,E. faecalis, mvaS mvaE mvaE mvaE mvaE mvaE E. faecium, E. faecium, E.faecium, E. faecium, E. faecium, mvaS mvaS mvaS mvaS mvaS E. gallinarum,L. grayi, E. faecium, E. gallinarum, E. casseliflavus, E. faecalis, mvaSmvaE mvaE mvaE mvaE mvaE E. gallinarum, E. gallinarum, E. gallinarum, E.gallinarum, E. gallinarum, mvaS mvaS mvaS mvaS mvaS E. casseliflavus, L.grayi, E. faecium, E. gallinarum, E. casseliflavus, E. faecalis, mvaSmvaE mvaE mvaE mvaE mvaE E. casseliflavus, E. casseliflavus, E.casseliflavus, E. casseliflavus, E. casseliflavus, mvaS mvaS mvaS mvaSmvaS E. faecalis, L. grayi, E. faecium, E. gallinarum, E. casseliflavus,E. faecalis, mvaS mvaE mvaE mvaE mvaE mvaE E. faecalis, E. faecalis, E.faecalis, E. faecalis, E. faecalis, mvaS mvaS mvaS mvaS mvaS

Exemplary MvaE Polypeptides and Nucleic Acids

The mvaE gene encodes a polypeptide that possesses both thiolase andHMG-CoA reductase activities. The thiolase activity of the polypeptideencoded by the mvaE gene converts acetyl-CoA to acetoacetyl-CoA whereasthe HMG-CoA reductase enzymatic activity of the polypeptide converts3-hydroxy-3-methylglutaryl-CoA to mevalonate. Exemplary MvaEpolypeptides and nucleic acids include naturally-occurring polypeptidesand nucleic acids from any of the source organisms described herein aswell as mutant polypeptides and nucleic acids derived from any of thesource organisms described herein that have at least one activity of aMvaE polypeptide.

Mutant MvaE polypeptides include those in which one or more amino acidresidues have undergone an amino acid substitution while retaining MvaEpolypeptide activity (i.e., the ability to convert acetyl-CoA toacetoacetyl-CoA as well as the ability to convert3-hydroxy-3-methylglutaryl-CoA to mevalonate). The amino acidsubstitutions can be conservative or non-conservative and suchsubstituted amino acid residues can or cannot be one encoded by thegenetic code. The standard twenty amino acid “alphabet” has been dividedinto chemical families based on similarity of their side chains. Thosefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a chemically similar side chain (i.e.,replacing an amino acid having a basic side chain with another aminoacid having a basic side chain). A “non-conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a chemically different side chain (i.e.,replacing an amino acid having a basic side chain with another aminoacid having an aromatic side chain).

Amino acid substitutions in the MvaE polypeptide can be introduced toimprove the functionality of the molecule. For example, amino acidsubstitutions that increase the binding affinity of the MvaE polypeptidefor its substrate, or that improve its ability to convert acetyl-CoA toacetoacetyl-CoA and/or the ability to convert3-hydroxy-3-methylglutaryl-CoA to mevalonate can be introduced into theMvaE polypeptide. In some aspects, the mutant MvaE polypeptides containone or more conservative amino acid substitutions.

In one aspect, mvaE proteins that are not degraded or less prone todegradation can be used for the production of mevalonate, isoprene,isoprenoid precursors, and/or isoprenoids. Examples of gene products ofmvaE genes that are not degraded or less prone to degradation which canbe used include, but are not limited to, those from the organisms E.faecium, E. gallinarum, E. casseliflavus, E. faecalis, and L. grayi. Oneof skill in the art can express mvaE protein in E. coli BL21 (DE3) andlook for absence of fragments by any standard molecular biologytechniques. For example, absence of fragments can be identified onSafestain stained SDS-PAGE gels following His-tag mediated purificationor when expressed in mevalonate, isoprene or isoprenoid producing E.coli BL21 using the methods of detection described herein.

Standard methods, such as those described in Hedl et al., (J. Bacteriol.2002, April; 184(8): 2116-2122) can be used to determine whether apolypeptide has mvaE activity, by measuring acetoacetyl-CoA thiolase aswell as HMG-CoA reductase activity. In an exemplary assay,acetoacetyl-CoA thiolase activity is measured by spectrophotometer tomonitor the change in absorbance at 302 nm that accompanies theformation or thiolysis of acetoacetyl-CoA. Standard assay conditions foreach reaction to determine synthesis of acetoacetyl-CoA are 1 mMacetyl-CoA, 10 mM MgCl₂, 50 mM Tris, pH 10.5 and the reaction isinitiated by addition of enzyme. Assays can employ a final volume of 200μL. For the assay, 1 enzyme unit (eu) represents the synthesis orthiolysis in 1 min of 1 μmol of acetoacetyl-CoA. In another exemplaryassay, of HMG-CoA reductase activity can be monitored byspectrophotometer by the appearance or disappearance of NADP(H) at 340nm. Standard assay conditions for each reaction measured to showreductive deacylation of HMG-CoA to mevalonate are 0.4 mM NADPH, 1.0 mM(R,S)-HMG-CoA, 100 mM KCl, and 100 mM K_(x)PO₄, pH 6.5. Assays employ afinal volume of 200 μL. Reactions are initiated by adding the enzyme.For the assay, 1 eu represents the turnover, in 1 min, of 1 μmol ofNADP(H). This corresponds to the turnover of 0.5 μmol of HMG-CoA ormevalonate.

Alternatively, production of mevalonate in cells (such as bacterial,fungal, or algal cells) can be measured by, without limitation, gaschromatography (see U.S. Patent Application Publication No.: US2005/0287655 A1) or HPLC (See U.S. patent application Ser. No.12/978,324). As an exemplary assay, cultures can be inoculated in shaketubes containing LB broth supplemented with one or more antibiotics andincubated for 14 h at 34° C. at 250 rpm. Next, cultures can be dilutedinto well plates containing TM3 media supplemented with 1% Glucose, 0.1%yeast extract, and 200 μM IPTG to final OD of 0.2. The plate are thensealed with a Breath Easier membrane (Diversified Biotech) and incubatedat 34° C. in a shaker/incubator at 600 rpm for 24 hours. 1 mL of eachculture is then centrifuged at 3,000×g for 5 min. Supernatant is thenadded to 20% sulfuric acid and incubated on ice for 5 min. The mixtureis then centrifuged for 5 min at 3000×g and the supernatant wascollected for HPLC analysis. The concentration of mevalonate in samplesis determined by comparison to a standard curve of mevalonate (Sigma).The glucose concentration can additionally be measured by performing aglucose oxidase assay according to any method known in the art. UsingHPLC, levels of mevalonate can be quantified by comparing the refractiveindex response of each sample versus a calibration curve generated byrunning various mevalonate containing solutions of known concentration.

Exemplary mvaE nucleic acids include nucleic acids that encode apolypeptide, fragment of a polypeptide, peptide, or fusion polypeptidethat has at least one activity of an MvaE polypeptide. Exemplary MvaEpolypeptides and nucleic acids include naturally-occurring polypeptidesand nucleic acids from any of the source organisms described herein aswell as mutant polypeptides and nucleic acids derived from any of thesource organisms described herein. Exemplary mvaE nucleic acids include,for example, mvaE nucleic acids isolated from Listeria grayi DSM 20601,Enterococcus faecium, Enterococcus gallinarum EG2, Enterococcuscasseliflavus and/or Enterococcus faecalis. The mvaE nucleic acidencoded by the Listeria grayi DSM 20601 mvaE gene can have at leastabout 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85% sequence identity to SEQ ID NO:1. In another aspect, the mvaEnucleic acid encoded by the Listeria grayi DSM20601 mvaE gene can haveat least about 84%, 83%, 82%, 81%, or 80% sequence identity to SEQ IDNO:1. The mvaE nucleic acid encoded by the Enterococcus faecium mvaEgene can have at least about 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, or 85% sequence identity to SEQ ID NO:3.In another aspect, the mvaE nucleic acid encoded by the Enterococcusfaecium mvaE gene can have at least about 84%, 83%, 82%, 81%, or 80%sequence identity to SEQ ID NO:3. The mvaE nucleic acid encoded by theEnterococcus gallinarum EG2 mvaE gene can have at least about 99%, 98%,97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85%sequence identity to SEQ ID NO:5. In another aspect, the mvaE nucleicacid encoded by the Enterococcus gallinarum EG2 mvaE gene can have atleast about 84%, 83%, 82%, 81%, or 80% sequence identity to SEQ ID NO:5.The mvaE nucleic acid encoded by the Enterococcus casseliflavus mvaEgene can have at least about 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, or 85% sequence identity to SEQ ID NO:7.In another aspect, the mvaE nucleic acid encoded by the Enterococcuscasseliflavus mvaE gene can have at least about 84%, 83%, 82%, 81%, or80% sequence identity to SEQ ID NO:7. The mvaE nucleic acid encoded bythe Enterococcus faecalis mvaE gene can have at least about 99%, 98%,97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85%sequence identity to SEQ ID NO:18. In any of the aspects herein, theupper MVA pathway polypeptides may be encoded by a nucleic acid with atleast about 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%,87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity to any oneof SEQ ID NOs:1-8 and 18-19. In any of the aspects herein, the upper MVApathway polypeptides may be encoded by a nucleic acid with of any one ofSEQ ID NOs: 1-8 and 18-19.

Exemplary MvaE polypeptides include fragments of a polypeptide, peptide,or fusion polypeptide that has at least one activity of an MvaEpolypeptide. Exemplary MvaE polypeptides and include naturally-occurringpolypeptides from any of the source organisms described herein as wellas mutant polypeptides derived from any of the source organismsdescribed herein. Exemplary MvaE polypeptides include, for example, MvaEpolypeptides isolated from Listeria grayi DSM20601, Enterococcusfaecium, Enterococcus gallinarum EG2, and/or Enterococcus casseliflavus.The MvaE polypeptide encoded by the Listeria grayi DSM 20601 mvaE genecan have at least about 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, 85% sequence identity to SEQ ID NO:11. Inanother aspect, the MvaE polypeptide encoded by the Listeria grayi DSM20601 mvaE gene can have at least about 84%, 83%, 82%, 81%, or 80%sequence identity to SEQ ID NO:11. The MvaE polypeptide encoded by theEnterococcus faecium mvaE gene can have at least about 99%, 98%, 97%,96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85% sequenceidentity to SEQ ID NO:13. In another aspect, the MvaE polypeptideencoded by the Enterococcus faecium mvaE gene can have at least about84%, 83%, 82%, 81%, or 80% sequence identity to SEQ ID NO:13. The MvaEpolypeptide encoded by the Enterococcus gallinarum EG2 mvaE gene canhave at least about 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%,89%, 88%, 87%, 86%, or 85% sequence identity to SEQ ID NO:9. In anotheraspect, the MvaE polypeptide encoded by the Enterococcus gallinarum EG2mvaE gene can have at least about 84%, 83%, 82%, 81%, or 80% sequenceidentity to SEQ ID NO:9. The MvaE polypeptide encoded by theEnterococcus casseliflavus mvaE gene can have at least about 99%, 98%,97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85%sequence identity to SEQ ID NO:15. In another aspect, the MvaEpolypeptide encoded by the Enterococcus casseliflavus mvaE gene can haveat least about 84%, 83%, 82%, 81%, or 80% sequence identity to SEQ IDNO:15. In any of the aspects herein, the upper MVA pathway polypeptidesmay be encoded by a polypeptide with at least about 99%, 98%, 97%, 96%,95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%,81%, or 80% sequence identity to any one of SEQ ID NOs:9-16 and 20-21.In any of the aspects herein, the upper MVA pathway polypeptides may beencoded by a polypeptide with any one of SEQ ID NOs:9-16 and 20-21.

The mvaE nucleic acid can be expressed in a cell (such as a bacterialcell) on a multicopy plasmid. The plasmid can be a high copy plasmid, alow copy plasmid, or a medium copy plasmid. Alternatively, the mvaEnucleic acid can be integrated into the host cell's chromosome. For bothheterologous expression of an mvaE nucleic acid on a plasmid or as anintegrated part of the host cell's chromosome, expression of the nucleicacid can be driven by either an inducible promoter or a constitutivelyexpressing promoter. The promoter can be a strong driver of expression,it can be a weak driver of expression, or it can be a medium driver ofexpression of the mvaE nucleic acid.

Exemplary MvaS Polypeptides and Nucleic Acids

The mvaS gene encodes a polypeptide that possesses HMG-CoA synthaseactivity. This polypeptide can convert acetoacetyl-CoA to3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Exemplary MvaS polypeptidesand nucleic acids include naturally-occurring polypeptides and nucleicacids from any of the source organisms described herein as well asmutant polypeptides and nucleic acids derived from any of the sourceorganisms described herein that have at least one activity of a MvaSpolypeptide.

Mutant MvaS polypeptides include those in which one or more amino acidresidues have undergone an amino acid substitution while retaining MvaSpolypeptide activity (i.e., the ability to convert acetoacetyl-CoA to3-hydroxy-3-methylglutaryl-CoA). Amino acid substitutions in the MvaSpolypeptide can be introduced to improve the functionality of themolecule. For example, amino acid substitutions that increase thebinding affinity of the MvaS polypeptide for its substrate, or thatimprove its ability to convert acetoacetyl-CoA to3-hydroxy-3-methylglutaryl-CoA can be introduced into the MvaSpolypeptide. In some aspects, the mutant MvaS polypeptides contain oneor more conservative amino acid substitutions.

Standard methods, such as those described in Quant et al. (Biochem J.,1989, 262:159-164), can be used to determine whether a polypeptide hasmvaS activity, by measuring HMG-CoA synthase activity. In an exemplaryassay, HMG-CoA synthase activity can be assayed byspectrophotometrically measuring the disappearance of the enol form ofacetoacetyl-CoA by monitoring the change of absorbance at 303 nm. Astandard 1 mL assay system containing 50 mm-Tris/HCl, pH 8.0, 10mM-MgCl₂ and 0.2 mM dithiothreitol at 30° C.; 5 mM-acetyl phosphate, 10,M-acetoacetyl-CoA and 5 ul samples of extracts can be added, followed bysimultaneous addition of acetyl-CoA (100 μM) and 10 units of PTA.HMG-CoA synthase activity is then measured as the difference in the ratebefore and after acetyl-CoA addition. The absorption coefficient ofacetoacetyl-CoA under the conditions used (pH 8.0, 10 mM-MgCl₂), is12.2×10³ M⁻¹ cm⁻¹. By definition, 1 unit of enzyme activity causes 1μmol of acetoacetyl-CoA to be transformed per minute.

Alternatively, production of mevalonate in cells (such as bacterial,fungal, or algal cells) can be measured by, without limitation, gaschromatography (see U.S. Patent Application Publication No.: US2005/0287655 A1) or HPLC (See U.S. patent application Ser. No.12/978,324). As an exemplary assay, cultures can be inoculated in shaketubes containing LB broth supplemented with one or more antibiotics andincubated for 14 h at 34° C. at 250 rpm. Next, cultures can be dilutedinto well plates containing TM3 media supplemented with 1% Glucose, 0.1%yeast extract, and 200 μM IPTG to final OD of 0.2. The plate are thensealed with a Breath Easier membrane (Diversified Biotech) and incubatedat 34° C. in a shaker/incubator at 600 rpm for 24 hours. 1 mL of eachculture is then centrifuged at 3,000×g for 5 min. Supernatant is thenadded to 20% sulfuric acid and incubated on ice for 5 min. The mixtureis then centrifuged for 5 min at 3000×g and the supernatant wascollected for HPLC analysis. The concentration of mevalonate in samplesis determined by comparison to a standard curve of mevalonate (Sigma).The glucose concentration can additionally be measured by performing aglucose oxidase assay according to any method known in the art. UsingHPLC, levels of mevalonate can be quantified by comparing the refractiveindex response of each sample versus a calibration curve generated byrunning various mevalonate containing solutions of known concentration.

Exemplary mvaS nucleic acids include nucleic acids that encode apolypeptide, fragment of a polypeptide, peptide, or fusion polypeptidethat has at least one activity of an MvaS polypeptide. Exemplary MvaSpolypeptides and nucleic acids include naturally-occurring polypeptidesand nucleic acids from any of the source organisms described herein aswell as mutant polypeptides and nucleic acids derived from any of thesource organisms described herein. Exemplary mvaS nucleic acids include,for example, mvaS nucleic acids isolated from Listeria grayi DSM 20601,Enterococcus faecium, Enterococcus gallinarum EG2, and/or Enterococcuscasseliflavus. The mvaS nucleic acid encoded by the Listeria grayi DSM20601 mvaS gene can have at least about 99%, 98%, 97%, 96%, 95%, 95%,93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85% sequence identity to SEQID NO:2. The mvaS nucleic acid encoded by the Listeria grayi DSM20601mvaS gene can also have at least about 84%, 83%, 82%, 81%, or 80%sequence identity to SEQ ID NO:2. The mvaS nucleic acid encoded by theEnterococcus faecium mvaS gene can have at least about 99%, 98%, 97%,96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85% sequenceidentity to SEQ ID NO:4. The mvaS nucleic acid encoded by theEnterococcus faecium mvaS gene can have at least about 84%, 83%, 82%,81%, or 80% sequence identity to SEQ ID NO:4. The mvaS nucleic acidencoded by the Enterococcus gallinarum EG2 mvaS gene can have at leastabout 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, or 85% sequence identity to SEQ ID NO:6. The mvaS nucleic acidencoded by the Enterococcus gallinarum EG2 mvaS gene can have at leastabout 84%, 83%, 82%, 81%, or 80% sequence identity to SEQ ID NO:6. ThemvaS nucleic acid encoded by the Enterococcus casseliflavus mvaS genecan have at least about 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, or 85% sequence identity to SEQ ID NO:8. ThemvaS nucleic acid encoded by the Enterococcus casseliflavus mvaS genecan have at least about 84%, 83%, 82%, 81%, or 80% sequence identity toSEQ ID NO:8.

Exemplary MvaS polypeptides include fragments of a polypeptide, peptide,or fusion polypeptide that has at least one activity of an MvaSpolypeptide. Exemplary MvaS polypeptides include naturally-occurringpolypeptides and polypeptides from any of the source organisms describedherein as well as mutant polypeptides derived from any of the sourceorganisms described herein. Exemplary MvaS polypeptides include, forexample, MvaS polypeptides isolated from Listeria grayi DSM 20601,Enterococcus faecium, Enterococcus gallinarum EG2, and/or Enterococcuscasseliflavus. The MvaS polypeptide encoded by the Listeria grayi DSM20601 mvaS gene can have at least about 99%, 98%, 97%, 96%, 95%, 95%,93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85% sequence identity to SEQID NO:12. The MvaS polypeptide encoded by the Listeria grayi DSM20601mvaS gene can also have at least about 84%, 83%, 82%, 81%, or 80%sequence identity to SEQ ID NO:12. The MvaS polypeptide encoded by theEnterococcus faecium mvaS gene can have at least about 99%, 98%, 97%,96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85% sequenceidentity to SEQ ID NO:14. The MvaS polypeptide encoded by theEnterococcus faecium mvaS gene can have at least about 84%, 83%, 82%,81%, or 80% sequence identity to SEQ ID NO:14. The MvaS polypeptideencoded by the Enterococcus gallinarum EG2 mvaS gene can have at leastabout 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, or 85% sequence identity to SEQ ID NO:10. The MvaS polypeptideencoded by the Enterococcus gallinarum EG2 mvaS gene can have at leastabout 84%, 83%, 82%, 81%, or 80% sequence identity to SEQ ID NO:10. TheMvaS polypeptide encoded by the Enterococcus casseliflavus mvaS gene canhave at least about 99%, 98%, 97%, 96%, 95%, 95%, 93%, 92%, 91%, 90%,89%, 88%, 87%, 86%, or 85% sequence identity to SEQ ID NO:16. The MvaSpolypeptide encoded by the Enterococcus casseliflavus mvaS gene can haveat least about 84%, 83%, 82%, 81%, or 80% sequence identity to SEQ IDNO:16.

The mvaS nucleic acid can be expressed in a cell (such as a bacterialcell) on a multicopy plasmid. The plasmid can be a high copy plasmid, alow copy plasmid, or a medium copy plasmid. Alternatively, the mvaSnucleic acid can be integrated into the host cell's chromosome. For bothheterologous expression of an mvaS nucleic acid on a plasmid or as anintegrated part of the host cell's chromosome, expression of the nucleicacid can be driven by either an inducible promoter or a constitutivelyexpressing promoter. The promoter can be a strong driver of expression,it can be a weak driver of expression, or it can be a medium driver ofexpression of the mvaS nucleic acid.

Nucleic Acids Encoding Acetoacetyl-CoA Synthase Polypeptides

In one aspect, any of the cells (such as bacterial, fungal, or algalcells) described herein can contain one or more heterologous nucleicacid(s) encoding an acetoacetyl-CoA synthase polypeptide. Theacetoacetyl-CoA synthase gene (also known as nphT7) is a gene encodingan enzyme having the activity of synthesizing acetoacetyl-CoA frommalonyl-CoA and acetyl-CoA and having minimal activity (e.g., noactivity) of synthesizing acetoacetyl-CoA from two acetyl-CoA molecules.See, e.g., Okamura et al., PNAS Vol 107, No. 25, pp. 11265-11270 (2010),the contents of which are expressly incorporated herein for teachingabout nphT7. An acetoacetyl-CoA synthase gene from an actinomycete ofthe genus Streptomyces CL190 strain was described in Japanese PatentPublication (Kokai) No. 2008-61506 A and U.S. Patent ApplicationPublication No. 2010/0285549, the disclosure of each of which areincorporated by reference herein. Acetoacetyl-CoA synthase can also bereferred to as acetyl-CoA:malonyl CoA acyltransferase. A representativeacetoacetyl-CoA synthase (or acetyl-CoA:malonyl CoA acyltransferase)that can be used is Genbank AB540131.1.

In one aspect, acetoacetyl-CoA synthase of the present inventionsynthesizes acetoacetyl-CoA from malonyl-CoA and acetyl-CoA via anirreversible reaction. The use of acetoacetyl-CoA synthase to generateacetyl-CoA provides an additional advantage in that this reaction isirreversible while acetoacetyl-CoA thiolase enzyme's action ofsynthesizing acetoacetyl-CoA from two acetyl-CoA molecules isreversible. Consequently, the use of acetoacetyl-CoA synthase tosynthesize acetoacetyl-CoA from malonyl-CoA and acetyl-CoA can result insignificant improvement in productivity for isoprene compared with usingthiolase to generate the end same product.

Furthermore, the use of acetoacetyl-CoA synthase to produce isopreneprovides another advantage in that acetoacetyl-CoA synthase can convertmalonyl CoA to acetyl-CoA via decarboxylation of the malonyl CoA. Thus,stores of starting substrate are not limited by the starting amounts ofacetyl-CoA. The synthesis of acetoacetyl-CoA by acetoacetyl-CoA synthasecan still occur when the starting substrate is only malonyl-CoA. In oneaspect, the pool of starting malonyl-CoA is increased by using hoststrains that have more malonyl-CoA. Such increased pools can benaturally occurring or be engineered by molecular manipulation. See, forexample Fowler, et al., Applied and Environmental Microbiology, Vol. 75,No. 18, pp. 5831-5839 (2009).

In any of the aspects or embodiments described herein, an enzyme thathas the ability to synthesize acetoacetyl-CoA from malonyl-CoA andacetyl-CoA can be used. Non-limiting examples of such an enzyme aredescribed herein. In certain embodiments described herein, anacetoacetyl-CoA synthase gene derived from an actinomycete of the genusStreptomyces having the activity of synthesizing acetoacetyl-CoA frommalonyl-CoA and acetyl-CoA can be used.

An example of such an acetoacetyl-CoA synthase gene is the gene encodinga protein having the amino acid sequence of SEQ ID NO: 17. Such aprotein having the amino acid sequence of SEQ ID NO: 17 corresponds toan acetoacetyl-CoA synthase having activity of synthesizingacetoacetyl-CoA from malonyl-CoA and acetyl-CoA and having no activityof synthesizing acetoacetyl-CoA from two acetyl-CoA molecules.

In one embodiment, the gene encoding a protein having the amino acidsequence of SEQ ID NO: 17 can be obtained by a nucleic acidamplification method (e.g., PCR) with the use of genomic DNA obtainedfrom an actinomycete of the Streptomyces sp. CL190 strain as a templateand a pair of primers that can be designed with reference to JapanesePatent Publication (Kokai) No. 2008-61506 A.

As described herein, an acetoacetyl-CoA synthase gene for use in thepresent invention is not limited to a gene encoding a protein having theamino acid sequence of SEQ ID NO: 17 from an actinomycete of theStreptomyces sp. CL190 strain. Any gene encoding a protein having theability to synthesize acetoacetyl-CoA from malonyl-CoA and acetyl-CoAand which does not synthesize acetoacetyl-CoA from two acetyl-CoAmolecules can be used in the presently described methods. In certainembodiments, the acetoacetyl-CoA synthase gene can be a gene encoding aprotein having an amino acid sequence with high similarity orsubstantially identical to the amino acid sequence of SEQ ID NO: 17 andhaving the function of synthesizing acetoacetyl-CoA from malonyl-CoA andacetyl-CoA. The expression “highly similar” or “substantially identical”refers to, for example, at least about 80% identity, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, and at least about 99% identity.As used above, the identity value corresponds to the percentage ofidentity between amino acid residues in a different amino acid sequenceand the amino acid sequence of SEQ ID NO: 17, which is calculated byperforming alignment of the amino acid sequence of SEQ ID NO: 17 and thedifferent amino acid sequence with the use of a program for searchingfor a sequence similarity.

In other embodiments, the acetoacetyl-CoA synthase gene may be a geneencoding a protein having an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 17 by substitution, deletion, addition, orinsertion of 1 or more amino acid(s) and having the function ofsynthesizing acetoacetyl-CoA from malonyl-CoA and acetyl-CoA. Herein,the expression “more amino acids” refers to, for example, 2 to 30 aminoacids, preferably 2 to 20 amino acids, more preferably 2 to 10 aminoacids, and most preferably 2 to 5 amino acids.

In still other embodiments, the acetoacetyl-CoA synthase gene mayconsist of a polynucleotide capable of hybridizing to a portion or theentirety of a polynucleotide having a nucleotide sequence complementaryto the nucleotide sequence encoding the amino acid sequence of SEQ IDNO: 17 under stringent conditions and capable of encoding a proteinhaving the function of synthesizing acetoacetyl-CoA from malonyl-CoA andacetyl-CoA. Herein, hybridization under stringent conditions correspondsto maintenance of binding under conditions of washing at 60° C. 2×SSC.Hybridization can be carried out by conventionally known methods such asthe method described in J. Sambrook et al. Molecular Cloning, ALaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001).

As described herein, a gene encoding an acetoacetyl-CoA synthase havingan amino acid sequence that differs from the amino acid sequence of SEQID NO: 17 can be isolated from potentially any organism, for example, anactinomycete that is not obtained from the Streptomyces sp. CL190strain. In addition, acetoacetyl-CoA synthase genes for use herein canbe obtained by modifying a polynucleotide encoding the amino acidsequence of SEQ ID NO: 17 by a method known in the art. Mutagenesis of anucleotide sequence can be carried out by a known method such as theKunkel method or the gapped duplex method or by a method similar toeither thereof. For instance, mutagenesis may be carried out with theuse of a mutagenesis kit (e.g., product names; Mutant-K and Mutant-G(TAKARA Bio)) for site-specific mutagenesis, product name; an LA PCR invitro Mutagenesis series kit (TAKARA Bio), and the like.

The activity of an acetoacetyl-CoA synthase having an amino acidsequence that differs from the amino acid sequence of SEQ ID NO: 17 canbe evaluated as described below. Specifically, a gene encoding a proteinto be evaluated is first introduced into a host cell such that the genecan be expressed therein, followed by purification of the protein by atechnique such as chromatography. Malonyl-CoA and acetyl-CoA are addedas substrates to a buffer containing the obtained protein to beevaluated, followed by, for example, incubation at a desired temperature(e.g., 10° C. to 60° C.). After the completion of reaction, the amountof substrate lost and/or the amount of product (acetoacetyl-CoA)produced are determined. Thus, it is possible to evaluate whether or notthe protein being tested has the function of synthesizingacetoacetyl-CoA from malonyl-CoA and acetyl-CoA and to evaluate thedegree of synthesis. In such case, it is possible to examine whether ornot the protein has the activity of synthesizing acetoacetyl-CoA fromtwo acetyl-CoA molecules by adding acetyl-CoA alone as a substrate to abuffer containing the obtained protein to be evaluated and determiningthe amount of substrate lost and/or the amount of product produced in asimilar manner.

Recombinant Microorganisms Capable of Increased Production of Mevalonate

The recombinant microorganisms (e.g., recombinant bacterial, fungal, oralgal cells) described herein have the ability to produce mevalonate atan amount and/or concentration greater than that of the same cellswithout any manipulation to the various genes or enzymatic pathwaysdescribed herein. The recombinant microorganisms (e.g., bacterial cells)that have been engineered for modulation in the various pathwaysdescribed herein to increase carbon flux to mevalonate can be used toproduce mevalonate. In some aspects, the cells contain one or morenucleic acids encoding one or more acetylating proteins, wherein saidcells have been engineered such that the expression of the nucleic acidsand/or activity of the acetylating protein(s) is modulated. Theacetylating proteins can be acetyltransferases (such as, but not limitedto, YfiQ) and/or deacetylases (such as, but not limited to CobB). Insome embodiments, the activity of the YfiQ polypeptide is modulated bydecreasing, attenuating, or deleting the expression of the gene encodingthe YfiQ polypeptide (such as, but not limited to, deletion of anendogenous yfiQ gene). In other embodiments, the activity of the CobBpolypeptide is modulated by increasing the expression of the geneencoding the CobB protein (such as, but not limited to, increasing theexpression of an endogenous cobB gene or heterologous expression of anucleic acid encoding cobB). In other aspects, culturing the recombinantcells described herein in a suitable media results in improvedproduction of mevalonate compared to a cell capable of producingmevalonate that does not comprise one or more acetylating proteinswherein said proteins are engineered such that their expression and/oractivity is modulated. In other embodiments, improved production ofmevalonate is characterized by one or more of an increase in mevalonatespecific productivity, an increase in mevalonate titer, an increase inmevalonate yield, an increase in cell viability, and/or a decrease inacetate production.

In one aspect, the recombinant cells (such as bacterial, fungal, oralgal cells) described herein which have been engineered such that theexpression of the nucleic acids and/or activity of the acetylatingprotein(s) is modulated to have the ability to produce mevalonate at aconcentration greater than that of the same cells which have not beenengineered such that the expression of the nucleic acids and/or activityof the acetylating protein(s) is modulated. The cells (such asbacterial, fungal, or algal cells) can produce greater than about 30mg/L/hr/OD, 40 mg/L/hr/OD, 50 mg/L/hr/OD, 60 mg/L/hr/OD, 70 mg/L/hr/OD,80 mg/L/hr/OD, 90 mg/L/hr/OD, 100 mg/L/hr/OD, 110 mg/L/hr/OD, 120mg/L/hr/OD, 130 mg/L/hr/OD, 140 mg/L/hr/OD, 150 mg/L/hr/OD, 160mg/L/hr/OD, 170 mg/L/hr/OD, 180 mg/L/hr/OD, 190 mg/L/hr/OD, or 200mg/L/hr/OD of mevalonate, inclusive, as well as any numerical value inbetween these numbers. In one exemplary embodiment, the cells canproduce greater than about 85 mg/L/hr/OD of mevalonate.

The host cells (such as bacterial, fungal, or algal cells) describedherein which have been engineered such that the expression of thenucleic acids and/or activity of the acetylating protein(s) is modulatedto have the ability to produce higher peak titers of mevalonate incomparison to that of the same cells which have not been engineered suchthat the expression of the nucleic acids and/or activity of theacetylating protein(s) is modulated. In another aspect, the cells (suchas bacterial, fungal, or algal cells) described herein producemevalonate at a higher peak titer than that of the same cells that havenot been modified to contain one or more acetylating proteins whereinsaid proteins are engineered such that their expression and/or activityis modulated when cultured in a suitable medium. The cells (such asbacterial, fungal, or algal cells) can produce greater than about 50g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L, 110 g/L, 120 g/L, 130 g/L,140 g/L, 150 g/L, 160 g/L, 170 g/L, 180 g/L, 190 g/L, 200 g/L, 210 g/L,220 g/L, 230 g/L, 240 g/L, 250 g/L, 260 g/L, 270 g/L, 280 g/L, 290 g/L,300 g/L peak titer of mevalonate after 48 hours of fermentation,inclusive, as well as any numerical value in between these numbers. Inone exemplary embodiment, the cells produce greater than about 105 g/Lpeak titer of mevalonate after 48 hours of fermentation.

The host cells (such as bacterial, fungal, or algal cells) describedherein which have been engineered such that the expression of thenucleic acids and/or activity of the acetylating protein(s) is modulatedto have the ability to produce a higher cell productivity index (CPI) incomparison to that of the same cells which have not been engineered suchthat the expression of the nucleic acids and/or activity of theacetylating protein(s) is modulated. In some aspects, the cells (such asbacterial, fungal, or algal cells) described herein have a higher CPIthan that of the same cells that have not been modified to contain oneor more acetylating proteins wherein said proteins are engineered suchthat their expression and/or activity is modulated. In one aspect, thecells can be cultured in minimal medium. The cells (such as bacterial,fungal, or algal cells) can have a CPI for mevalonate of at least about1 (g/g), 2 (g/g), 3 (g/g), 4 (g/g), 5 (g/g), 6 (g/g), 7 (g/g), 8 (g/g),9 (g/g), 10 (g/g), 11 (g/g), 12 (g/g), 13 (g/g), 14 (g/g), 15 (g/g), 20(g/g), 25 (g/g), or 30 (g/g) inclusive, as well as any numerical valuein between these numbers. In one exemplary embodiment, the cells have aCPI for mevalonate of at least about 4.5 (g/g).

The host cells (such as bacterial, fungal, or algal cells) describedherein which have been engineered such that the expression of thenucleic acids and/or activity of the acetylating protein(s) is modulatedto have the ability to produce higher mass yield of mevalonate incomparison to that of the same cells which have not been engineered suchthat the expression of the nucleic acids and/or activity of theacetylating protein(s) is modulated. In some aspects, the cells (such asbacterial, fungal, or algal cells) described herein have a higher massyield of mevalonate from glucose than that of the same cells that havenot been modified to contain one or more acetylating proteins whereinsaid proteins are engineered such that their expression and/or activityis modulated. In one aspect, the cells can be cultured in minimalmedium. The cells (such as bacterial, fungal, or algal cells) canproduce a mass yield of mevalonate from glucose of at least about 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or 55%,inclusive, as well as any numerical value in between these numbers. Inone exemplary embodiment, the cells produce a mass yield of mevalonatefrom glucose of at least about 38%.

Methods of Using Recombinant Cells to Produce High Amounts of Mevalonate

Also provided herein are methods for the production of mevalonate. Insome aspects, the method for producing mevalonate comprises: (a)culturing a composition comprising recombinant cells (such as bacterial,fungal, or algal cells) which have been modified to comprise one or moreacetylating proteins wherein said proteins are engineered such thattheir expression and/or activity is modulated as described herein(including any of the cells, such as the bacterial cells describedabove), or progeny thereof, capable of producing mevalonate; and (b)producing mevalonate. In some aspects, the method of producingmevalonate comprises the steps of culturing any of the recombinant cellsdescribed herein under conditions suitable for the production ofmevalonate and allowing the recombinant cells to produce mevalonate. Insome aspects, the method of producing mevalonate further comprises astep of recovering the mevalonate.

The method of producing mevalonate can also comprise the steps of: (a)culturing cells heterologously expressing one or more acetylatingproteins wherein said proteins are engineered such that their expressionand/or activity is modulated; and (b) producing mevalonate.Additionally, the cells can produce mevalonate in concentrations greaterthan that of the same cells lacking one or more acetylating proteinswherein said proteins are not engineered such that their expressionand/or activity is modulated.

Mevalonate can be produced in amounts greater than about 30 mg/L/hr/OD,40 mg/L/hr/OD, 50 mg/L/hr/OD, 60 mg/L/hr/OD, 70 mg/L/hr/OD, 80mg/L/hr/OD, 90 mg/L/hr/OD, 100 mg/L/hr/OD, 110 mg/L/hr/OD, 120mg/L/hr/OD, 130 mg/L/hr/OD, 140 mg/L/hr/OD, 150 mg/L/hr/OD, 160mg/L/hr/OD, 170 mg/L/hr/OD, 180 mg/L/hr/OD, 190 mg/L/hr/OD, or 200mg/L/hr/OD of mevalonate, inclusive, as well as any numerical value inbetween these numbers. In some aspects, the method of producingmevalonate further comprises a step of recovering the mevalonate. In oneexemplary embodiment, the instant methods for the production ofmevalonate can produce greater than about 85 mg/L/hr/OD of mevalonate.

The method of producing mevalonate can similarly comprise the steps of:(a) culturing cells which have been engineered for increased carbon fluxto mevalonate as described herein, wherein the cells heterologouslyexpress one or more acetylating proteins wherein said proteins areengineered such that their expression and/or activity is modulated; and(b) producing mevalonate, wherein the cells produce mevalonate with ahigher peak titer after hours of fermentation than that of the samecells lacking one or more acetylating proteins wherein said proteins areengineered such that their expression and/or activity is modulated.

The cells provided herein (such as bacterial, fungal, or algal cells)can produce greater than about 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L,100 g/L, 110 g/L, 120 g/L, 130 g/L, 140 g/L, 150 g/L, 160 g/L, 170 g/L,180 g/L, 190 g/L, or 200 g/L peak titer of mevalonate after 48 hours offermentation, inclusive, as well as any numerical value in between thesenumbers. In some aspects, the method of producing mevalonate furthercomprises a step of recovering the mevalonate. In one exemplaryembodiment, the instant methods for the production of mevalonate canproduce greater than about 105 g/L peak titer of mevalonate after 48hours of fermentation.

The method of producing mevalonate can similarly comprise the steps of:(a) culturing cells which have been engineered for increased carbon fluxto mevalonate as described herein, wherein the cells comprise one ormore acetylating proteins wherein said proteins are engineered such thattheir expression and/or activity is modulated; and (b) producingmevalonate, wherein the cells have a CPI for mevalonate higher than thatof the same cells lacking one or more copies of an upper MVA pathwaygene encoding one or more upper MVA pathway polypeptides, and which havenot been engineered for increased carbon flux to mevalonate production.In one exemplary embodiment, the instant methods for the production ofmevalonate can produce mevalonate using cells with a CPI for mevalonateof at least 4.5 (g/g). Alternatively, the cells (such as bacterial,fungal, or algal cells) can have a CPI of at least 1 (g/g), 2 (g/g), 3(g/g), 4 (g/g), 5 (g/g), 6 (g/g), 7 (g/g), 8 (g/g), 9 (g/g), 10 (g/g),11 (g/g), 12 (g/g), 13 (g/g), 14 (g/g), 15 (g/g), 20 (g/g), 25 (g/g), or30 (g/g) inclusive, as well as any numerical value in between thesenumbers. In some aspects, the method of producing mevalonate furthercomprises a step of recovering the mevalonate.

Provided herein are methods of using any of the cells described abovefor enhanced mevalonate production. The production of mevalonate by thecells can be enhanced by the expression of one or more acetylatingproteins wherein said proteins are engineered such that their expressionand/or activity is modulated. The production of mevalonate can beenhanced by about 1,000,000 folds (e.g., about 1 to about 500,000 folds,about 1 to about 50,000 folds, about 1 to about 5,000 folds, about 1 toabout 1,000 folds, about 1 to about 500 folds, about 1 to about 100folds, about 1 to about 50 folds, about 5 to about 100,000 folds, about5 to about 10,000 folds, about 5 to about 1,000 folds, about 5 to about500 folds, about 5 to about 100 folds, about 10 to about 50,000 folds,about 50 to about 10,000 folds, about 100 to about 5,000 folds, about200 to about 1,000 folds, about 50 to about 500 folds, or about 50 toabout 200 folds) compared to the production of mevalonate by cellswithout the expression of one or more acetylating proteins wherein saidproteins are engineered such that their expression and/or activity ismodulated.

Recombinant Cells Capable of Production of Isoprene

Isoprene (2-methyl-1,3-butadiene) is an important organic compound usedin a wide array of applications. For instance, isoprene is employed asan intermediate or a starting material in the synthesis of numerouschemical compositions and polymers, including in the production ofsynthetic rubber. Isoprene is also an important biological material thatis synthesized naturally by many plants and animals.

Isoprene is produced from DMAPP by the enzymatic action of isoprenesynthase. Therefore, without being bound to theory, it is thought thatincreasing the cellular production of mevalonate in cells (such asbacterial, fungal, or algal cells) by any of the compositions andmethods described above will similarly result in the production ofhigher amounts of isoprene. Increasing the molar yield of mevalonateproduction from glucose translates into higher molar yields ofisoprenoid precursors and isoprenoids, including isoprene, produced fromglucose when combined with appropriate enzymatic activity levels ofmevalonate kinase, phosphomevalonate kinase, diphosphomevalonatedecarboxylase, isopentenyl diphosphate isomerase and other appropriateenzymes for isoprene and isoprenoid production.

Production of isoprene can be made by using any of the recombinant hostcells described here where one or more of the enzymatic pathways havebeen manipulated such that enzyme activity is modulated to increasecarbon flow towards isoprene production. The recombinant microorganismsdescribed herein that have various enzymatic pathways manipulated forincreased carbon flow to mevalonate production can be used to produceisoprene. Any of the recombinant host cells expressing one or moreacetylating proteins (wherein said proteins are engineered such thattheir expression and/or activity is modulated) capable of increasedproduction of mevalonate described above can also be capable ofincreased production of isoprene. In some aspects, these cells furthercomprise one or more heterologous nucleic acids encoding polypeptides ofthe entire MVA pathway and a heterologous nucleic acid encoding anisoprene synthase polypeptide or a polypeptide having isoprene synthaseactivity. In other aspects, these cells further comprise one or moreheterologous nucleic acids encoding a phosphoketolase polypeptide.

Nucleic Acids Encoding Polypeptides of the Lower MVA Pathway

In some aspects of the invention, the cells described in any of thecells or methods described herein further comprise one or more nucleicacids encoding a lower mevalonate (MVA) pathway polypeptide(s). In someaspects, the lower MVA pathway polypeptide is an endogenous polypeptide.In some aspects, the endogenous nucleic acid encoding a lower MVApathway polypeptide is operably linked to a constitutive promoter. Insome aspects, the endogenous nucleic acid encoding a lower MVA pathwaypolypeptide is operably linked to an inducible promoter. In someaspects, the endogenous nucleic acid encoding a lower MVA pathwaypolypeptide is operably linked to a strong promoter. In a particularaspect, the cells are engineered to over-express the endogenous lowerMVA pathway polypeptide relative to wild-type cells. In some aspects,the endogenous nucleic acid encoding a lower MVA pathway polypeptide isoperably linked to a weak promoter.

The lower mevalonate biosynthetic pathway comprises mevalonate kinase(MVK), phosphomevalonate kinase (PMK), and diphosphomevalontedecarboxylase (MVD). In some aspects, the lower MVA pathway can furthercomprise isopentenyl diphosphate isomerase (IDI). Cells provided hereincan comprise at least one nucleic acid encoding isoprene synthase, oneor more upper MVA pathway polypeptides, and/or one or more lower MVApathway polypeptides. Polypeptides of the lower MVA pathway can be anyenzyme (a) that phosphorylates mevalonate to mevalonate 5-phosphate; (b)that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; and(c) that converts mevalonate 5-pyrophosphate to isopentenylpyrophosphate. More particularly, the enzyme that phosphorylatesmevalonate to mevalonate 5-phosphate can be from the group consisting ofM. mazei mevalonate kinase polypeptide, Lactobacillus mevalonate kinasepolypeptide, M. burtonii mevalonate kinase polypeptide, Lactobacillussakei mevalonate kinase polypeptide, yeast mevalonate kinasepolypeptide, Saccharomyces cerevisiae mevalonate kinase polypeptide,Streptococcus mevalonate kinase polypeptide, Streptococcus pneumoniaemevalonate kinase polypeptide, Streptomyces mevalonate kinasepolypeptide, and Streptomyces CL190 mevalonate kinase polypeptide. Inanother aspect, the enzyme that phosphorylates mevalonate to mevalonate5-phosphate is M. mazei mevalonate kinase.

In some aspects, the lower MVA pathway polypeptide is a heterologouspolypeptide. In some aspects, the cells comprise more than one copy of aheterologous nucleic acid encoding a lower MVA pathway polypeptide. Insome aspects, the heterologous nucleic acid encoding a lower MVA pathwaypolypeptide is operably linked to a constitutive promoter. In someaspects, the heterologous nucleic acid encoding a lower MVA pathwaypolypeptide is operably linked to an inducible promoter. In someaspects, the heterologous nucleic acid encoding a lower MVA pathwaypolypeptide is operably linked to a strong promoter. In some aspects,the heterologous nucleic acid encoding a lower MVA pathway polypeptideis operably linked to a weak promoter. In some aspects, the heterologouslower MVA pathway polypeptide is a polypeptide from Saccharomycescerevisiae, Enterococcus faecalis, or Methanosarcina mazei.

The nucleic acids encoding a lower MVA pathway polypeptide(s) can beintegrated into a genome of the cells or can be stably expressed in thecells. The nucleic acids encoding a lower MVA pathway polypeptide(s) canadditionally be on a vector.

Exemplary lower MVA pathway polypeptides are also provided below: (i)mevalonate kinase (MVK); (ii) phosphomevalonate kinase (PMK); (iii)diphosphomevalonate decarboxylase (MVD); and (iv) isopentenyldiphosphate isomerase (IDI). In particular, the lower MVK polypeptidecan be from the genus Methanosarcina and, more specifically, the lowerMVK polypeptide can be from Methanosarcina mazei. In other aspects, thelower MVK polypeptide can be from M. burtonii. Additional examples oflower MVA pathway polypeptides can be found in U.S. Patent ApplicationPublication 2010/0086978 the contents of which are expresslyincorporated herein by reference in their entirety with respect to lowerMVK pathway polypeptides and lower MVK pathway polypeptide variants.

Anyone of the cells described herein can comprise IDI nucleic acid(s)(e.g., endogenous or heterologous nucleic acid(s) encoding IDI).Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphatedelta-isomerase or IDI) catalyzes the interconversion of isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (e.g.,converting IPP into DMAPP and/or converting DMAPP into IPP). ExemplaryIDI polypeptides include polypeptides, fragments of polypeptides,peptides, and fusions polypeptides that have at least one activity of anIDI polypeptide. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has IDI polypeptide activityby measuring the ability of the polypeptide to interconvert IPP andDMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI nucleicacids include nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of an IDI polypeptide. Exemplary IDI polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Lower MVA pathway polypeptides include polypeptides, fragments ofpolypeptides, peptides, and fusions polypeptides that have at least oneactivity of a lower MVA pathway polypeptide. Exemplary lower MVA pathwaynucleic acids include nucleic acids that encode a polypeptide, fragmentof a polypeptide, peptide, or fusion polypeptide that has at least oneactivity of a lower MVA pathway polypeptide. Exemplary lower MVA pathwaypolypeptides and nucleic acids include naturally-occurring polypeptidesand nucleic acids from any of the source organisms described herein. Inaddition, variants of lower MVA pathway polypeptides that confer theresult of better isoprene production can also be used as well.

In some aspects, the lower MVA pathway polypeptide is a polypeptide fromSaccharomyces cerevisiae, Enterococcus faecalis, or Methanosarcinamazei. In some aspects, the MVK polypeptide is selected from the groupconsisting of Lactobacillus mevalonate kinase polypeptide, Lactobacillussakei mevalonate kinase polypeptide, yeast mevalonate kinasepolypeptide, Saccharomyces cerevisiae mevalonate kinase polypeptide,Streptococcus mevalonate kinase polypeptide, Streptococcus pneumoniaemevalonate kinase polypeptide, Streptomyces mevalonate kinasepolypeptide, Streptomyces CL190 mevalonate kinase polypeptide, M.burtonii mevalonate kinase polypeptide, and Methanosarcina mazeimevalonate kinase polypeptide. Any one of the promoters described herein(e.g., promoters described herein and identified in the Examples of thepresent disclosure including inducible promoters and constitutivepromoters) can be used to drive expression of any of the MVApolypeptides described herein.

Nucleic Acids Encoding Isoprene Synthase Polypeptides

In some aspects of the invention, the cells described in any of thecompositions or methods described herein (including host cells that havebeen engineered for increased carbon flux as described herein) furthercomprise one or more nucleic acids encoding an isoprene synthasepolypeptide or a polypeptide having isoprene synthase activity. In someaspects, the isoprene synthase polypeptide is an endogenous polypeptide.In some aspects, the endogenous nucleic acid encoding an isoprenesynthase polypeptide or a polypeptide having isoprene synthase activityis operably linked to a constitutive promoter. In some aspects, theendogenous nucleic acid encoding an isoprene synthase polypeptide or apolypeptide having isoprene synthase activity is operably linked to aninducible promoter. In some aspects, the endogenous nucleic acidencoding an isoprene synthase polypeptide or a polypeptide havingisoprene synthase activity is operably linked to a strong promoter. In aparticular aspect, the cells are engineered to over-express theendogenous isoprene synthase pathway polypeptide relative to wild-typecells. In some aspects, the endogenous nucleic acid encoding an isoprenesynthase polypeptide or a polypeptide having isoprene synthase activityis operably linked to a weak promoter. In some aspects, the isoprenesynthase polypeptide or a polypeptide having isoprene synthase activityis a polypeptide from Pueraria or Populus or a hybrid such as Populusalba x Populus tremula.

In some aspects, the isoprene synthase polypeptide is a heterologouspolypeptide. In some aspects, the cells comprise more than one copy of aheterologous nucleic acid encoding an isoprene synthase polypeptide. Insome aspects, the heterologous nucleic acid encoding an isoprenesynthase polypeptide is operably linked to a constitutive promoter. Insome aspects, the heterologous nucleic acid encoding an isoprenesynthase polypeptide is operably linked to an inducible promoter. Insome aspects, the heterologous nucleic acid encoding an isoprenesynthase polypeptide is operably linked to a strong promoter. In someaspects, the heterologous nucleic acid encoding an isoprene synthasepolypeptide is operably linked to a weak promoter.

The nucleic acids encoding an isoprene synthase polypeptide(s) can beintegrated into a genome of the host cells or can be stably expressed inthe cells. The nucleic acids encoding an isoprene synthasepolypeptide(s) can additionally be on a vector.

Exemplary isoprene synthase nucleic acids include nucleic acids thatencode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an isoprene synthasepolypeptide. Isoprene synthase polypeptides convert dimethylallyldiphosphate (DMAPP) into isoprene. Exemplary isoprene synthasepolypeptides include polypeptides, fragments of polypeptides, peptides,and fusions polypeptides that have at least one activity of an isoprenesynthase polypeptide. Exemplary isoprene synthase polypeptides andnucleic acids include naturally-occurring polypeptides and nucleic acidsfrom any of the source organisms described herein. In addition, variantsof isoprene synthase can possess improved activity such as improvedenzymatic activity. In some aspects, an isoprene synthase variant hasother improved properties, such as improved stability (e.g.,thermo-stability), and/or improved solubility.

Standard methods can be used to determine whether a polypeptide hasisoprene synthase polypeptide activity by measuring the ability of thepolypeptide to convert DMAPP into isoprene in vitro, in a cell extract,or in vivo. Isoprene synthase polypeptide activity in the cell extractcan be measured, for example, as described in Silver et al., J. Biol.Chem. 270:13010-13016, 1995. In one exemplary assay, DMAPP (Sigma) canbe evaporated to dryness under a stream of nitrogen and rehydrated to aconcentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 andstored at −20° C. To perform the assay, a solution of 5 μL of 1M MgCl₂,1 mM (250 μg/mL) DMAPP, 65 μL of Plant Extract Buffer (PEB) (50 mMTris-HCl, pH 8.0, 20 mM MgCl₂, 5% glycerol, and 2 mM DTT) can be addedto 25 μL of cell extract in a 20 mL Headspace vial with a metal screwcap and teflon coated silicon septum (Agilent Technologies) and culturedat 37° C. for 15 minutes with shaking. The reaction can be quenched byadding 200 μL of 250 mM EDTA and quantified by GC/MS.

In some aspects, the isoprene synthase polypeptide is a plant isoprenesynthase polypeptide or a variant thereof. In some aspects, the isoprenesynthase polypeptide is an isoprene synthase from Pueraria or a variantthereof. In some aspects, the isoprene synthase polypeptide is anisoprene synthase from Populus or a variant thereof. In some aspects,the isoprene synthase polypeptide is a poplar isoprene synthasepolypeptide or a variant thereof. In some aspects, the isoprene synthasepolypeptide is a kudzu isoprene synthase polypeptide or a variantthereof. In some aspects, the isoprene synthase polypeptide is apolypeptide from Pueraria or Populus or a hybrid, Populus alba x Populustremula, or a variant thereof.

In some aspects, the isoprene synthase polypeptide, the polypeptidehaving isoprene synthase activity or the corresponding nucleic acid isfrom the family Fabaceae, such as the Faboideae subfamily. In someaspects, the isoprene synthase polypeptide, the polypeptide havingisoprene synthase activity or the corresponding nucleic acid is fromPueraria montana (kudzu) (Sharkey et al., Plant Physiology 137: 700-712,2005), Pueraria lobata, poplar (such as Populus alba, Populus nigra,Populus trichocarpa, or Populus alba x tremula (CAC35696) (Miller etal., Planta 213: 483-487, 2001), aspen (such as Populus tremuloides)(Silver et al., JBC 270(22): 13010-1316, 1995), English Oak (Quercusrobur) (Zimmer et al., WO 98/02550), or a variant thereof. In someaspects, the isoprene synthase polypeptide, the polypeptide havingisoprene synthase activity or the corresponding nucleic acid is fromPueraria montana, Pueraria lobata, Populus tremuloides, Populus alba,Populus nigra, or Populus trichocarpa or a variant thereof. In someaspects, the isoprene synthase polypeptide, the polypeptide havingisoprene synthase activity or the corresponding nucleic acid is fromPopulus alba or a variant thereof. In some aspects, the nucleic acidencoding the isoprene synthase (e.g., isoprene synthase from Populusalba or a variant thereof) is codon optimized.

In some aspects, the isoprene synthase nucleic acid or polypeptide is anaturally-occurring polypeptide or nucleic acid (e.g.,naturally-occurring polypeptide or nucleic acid from Populus). In someaspects, the isoprene synthase nucleic acid or polypeptide is not awild-type or naturally-occurring polypeptide or nucleic acid. In someaspects, the isoprene synthase nucleic acid or polypeptide is a variantof a wild-type or naturally-occurring polypeptide or nucleic acid (e.g.,a variant of a wild-type or naturally-occurring polypeptide or nucleicacid from Populus).

In some aspects, the isoprene synthase polypeptide is a variant. In someaspects, the isoprene synthase polypeptide is a variant of a wild-typeor naturally occurring isoprene synthase. In some aspects, the varianthas improved activity such as improved catalytic activity compared tothe wild-type or naturally occurring isoprene synthase. The increase inactivity (e.g., catalytic activity) can be at least about any of 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some aspects, theincrease in activity such as catalytic activity is at least about any of1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 30 folds, 40 folds, 50folds, 75 folds, or 100 folds. In some aspects, the increase in activitysuch as catalytic activity is about 10% to about 100 folds (e.g., about20% to about 100 folds, about 50% to about 50 folds, about 1 fold toabout 25 folds, about 2 folds to about 20 folds, or about 5 folds toabout 20 folds). In some aspects, the variant has improved solubilitycompared to the wild-type or naturally occurring isoprene synthase. Theincrease in solubility can be at least about any of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 95%. The increase in solubility can be atleast about any of 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 30folds, 40 folds, 50 folds, 75 folds, or 100 folds. In some aspects, theincrease in solubility is about 10% to about 100 folds (e.g., about 20%to about 100 folds, about 50% to about 50 folds, about 1 fold to about25 folds, about 2 folds to about 20 folds, or about 5 folds to about 20folds). In some aspects, the isoprene synthase polypeptide is a variantof naturally occurring isoprene synthase and has improved stability(such as thermo-stability) compared to the naturally occurring isoprenesynthase.

In some aspects, the variant has at least about 10%, at least about 20%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 100%, at least about 110%, at least about 120%, at leastabout 130%, at least about 140%, at least about 150%, at least about160%, at least about 170%, at least about 180%, at least about 190%, orat least about 200% of the activity of a wild-type or naturallyoccurring isoprene synthase. The variant can share sequence similaritywith a wild-type or naturally occurring isoprene synthase. In someaspects, a variant of a wild-type or naturally occurring isoprenesynthase can have at least about any of 40%, 50%, 60%, 70%, 75%, 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% aminoacid sequence identity as that of the wild-type or naturally occurringisoprene synthase. In some aspects, a variant of a wild-type ornaturally occurring isoprene synthase has any of about 70% to about99.9%, about 75% to about 99%, about 80% to about 98%, about 85% toabout 97%, or about 90% to about 95% amino acid sequence identity asthat of the wild-type or naturally occurring isoprene synthase.

In some aspects, the variant comprises a mutation in the wild-type ornaturally occurring isoprene synthase. In some aspects, the variant hasat least one amino acid substitution, at least one amino acid insertion,and/or at least one amino acid deletion. In some aspects, the varianthas at least one amino acid substitution. In some aspects, the number ofdiffering amino acid residues between the variant and wild-type ornaturally occurring isoprene synthase can be one or more, e.g. 1, 2, 3,4, 5, 10, 15, 20, 30, 40, 50, or more amino acid residues. Naturallyoccurring isoprene synthases can include any isoprene synthases fromplants, for example, kudzu isoprene synthases, poplar isoprenesynthases, English oak isoprene synthases, and willow isoprenesynthases. In some aspects, the variant is a variant of isoprenesynthase from Populus alba. In some aspects, the variant of isoprenesynthase from Populus alba has at least one amino acid substitution, atleast one amino acid insertion, and/or at least one amino acid deletion.In some aspects, the variant is a truncated Populus alba isoprenesynthase. In some aspects, the nucleic acid encoding variant (e.g.,variant of isoprene synthase from Populus alba) is codon optimized (forexample, codon optimized based on host cells where the heterologousisoprene synthase is expressed). In other aspects, the variant ofisoprene synthase from Populus alba has at least one amino acidsubstitution, at least one amino acid insertion, and/or at least oneamino acid deletion at the amino acid residue shown in Table 2. Inanother aspect, the variant of isoprene synthase comprises at least oneamino acid substitution, at least one amino acid deletion, or at leastone amino acid insertion at any of the amino acid residues shown inTable 2, wherein the amino acid residue numbering corresponds to theamino acid residue number of MEA P. alba isoprene synthase (SEQ ID NO:24). In one aspect, the P. alba isoprene synthase is a truncatedisoprene synthase, for example, MEA isoprene synthase which is 16 aminoacids shorter than full-length isoprene synthase.

TABLE 2 Isoprene Synthase Variants of P. Alba (MEA) A118E E472R S510CD323Y W392S S22K K463F S510V D323D W392T S21R K463T I342I G99D W392VS22K R71K K348F K161K A118P S22R R71L K348Y W392A A118Q E58L R71M K348KW392C A118A T481V R71V C437L W392F E41M T481Y R71R T240C S288Y G111ST502F K393L M460M M228Y S74Q T381L F542L R461A A3T S74S T381M P538KH424P W392Y K36D T381Y P538R H424H W392W S282H T383H P538P A448L F89DS282I T383L A503A A448Q F89E S282W E480I L436I A448V F89F S282Y E480RL436Y G389D E41Y S282S K393V L436F S444E E41E K36S K393I E488L S444SR43E K36T E415H E488M H511Y R43L K36W E415V E488T H511H K36E K36Y E415YE488W R071I K36H K36K R71H E488E R071K K36N R71I I342Y R071L K36P E58YC437M K374Y K36Q E135G C437W K374K A453I A363L C437Y L526E A453V K374YC437C L526Q A453A T381I M460A L526L V409I L436L I447T R242G V409T H254RI447V R242R K161C H254C I447Y A443G K161E E488C S444D A443Q K161N E488FG389E A443R K161Q T383Y L376I A443S G99E K414I L376M S13S G99G K414RL376L V268I S288A K414S I504F V268V S288C K414W I504I K161A S288T E472CE467H V409V W392I E472L E467W D323F W392M

In one embodiment, the MEA P. alba isoprene synthase is truncated sothat it is 16 amino acids shorter than full length P. alba isoprenesynthase.

The isoprene synthase polypeptide provided herein can be any of theisoprene synthases or isoprene synthase variants described in WO2009/132220, WO 2010/124146, and U.S. Patent Application PublicationNo.: 2010/0086978, the contents of which are expressly incorporatedherein by reference in their entirety with respect to the isoprenesynthases and isoprene synthase variants.

Anyone of the promoters described herein (e.g., promoters describedherein and identified in the Examples of the present disclosureincluding inducible promoters and constitutive promoters) can be used todrive expression of any of the isoprene synthases described herein.

Suitable isoprene synthases include, but are not limited to, thoseidentified by Genbank Accession Nos. AY341431, AY316691, AY279379,AJ457070, and AY182241. Types of isoprene synthases which can be used inany one of the compositions or methods including methods of makingmicroorganisms encoding isoprene synthase described herein are alsodescribed in International Patent Application Publication Nos.WO2009/076676, WO2010/003007, WO2009/132220, WO2010/031062,WO2010/031068, WO2010/031076, WO2010/013077, WO2010/031079,WO2010/148150, WO2010/124146, WO2010/078457, WO2010/148256, and WO2013/166320.

Nucleic Acids Encoding DXP Pathway Polypeptides

In some aspects of the invention, the cells described in any of thecompositions or methods described herein (including host cells that havebeen engineered for increased carbon flux as described herein) furthercomprise one or more heterologous nucleic acids encoding a DXSpolypeptide or other DXP pathway polypeptides. In some aspects, thecells further comprise a chromosomal copy of an endogenous nucleic acidencoding a DXS polypeptide or other DXP pathway polypeptides. In someaspects, the E. coli cells further comprise one or more nucleic acidsencoding an IDI polypeptide and a DXS polypeptide or other DXP pathwaypolypeptides. In some aspects, one nucleic acid encodes the isoprenesynthase polypeptide, IDI polypeptide, and DXS polypeptide or other DXPpathway polypeptides. In some aspects, one plasmid encodes the isoprenesynthase polypeptide, IDI polypeptide, and DXS polypeptide or other DXPpathway polypeptides. In some aspects, multiple plasmids encode theisoprene synthase polypeptide, IDI polypeptide, and DXS polypeptide orother DXP pathway polypeptides.

Exemplary DXS polypeptides include polypeptides, fragments ofpolypeptides, peptides, and fusions polypeptides that have at least oneactivity of a DXS polypeptide. Standard methods (such as those describedherein) can be used to determine whether a polypeptide has DXSpolypeptide activity by measuring the ability of the polypeptide toconvert pyruvate and D-glyceraldehyde-3-phosphate into1-deoxy-D-xylulose-5-phosphate in vitro, in a cell extract, or in vivo.Exemplary DXS polypeptides and nucleic acids and methods of measuringDXS activity are described in more detail in International PublicationNo. WO 2009/076676, U.S. patent application Ser. No. 12/335,071 (USPubl. No. 2009/0203102), WO 2010/003007, US Publ. No. 2010/0048964, WO2009/132220, and US Publ. No. 2010/0003716.

Exemplary DXP pathways polypeptides include, but are not limited to anyof the following polypeptides: DXS polypeptides, DXR polypeptides, MCTpolypeptides, CMK polypeptides, MCS polypeptides, HDS polypeptides, HDRpolypeptides, and polypeptides (e.g., fusion polypeptides) having anactivity of one, two, or more of the DXP pathway polypeptides. Inparticular, DXP pathway polypeptides include polypeptides, fragments ofpolypeptides, peptides, and fusions polypeptides that have at least oneactivity of a DXP pathway polypeptide. Exemplary DXP pathway nucleicacids include nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of a DXP pathway polypeptide. Exemplary DXP pathwaypolypeptides and nucleic acids include naturally-occurring polypeptidesand nucleic acids from any of the source organisms described herein aswell as mutant polypeptides and nucleic acids derived from any of thesource organisms described herein. Exemplary DXP pathway polypeptidesand nucleic acids and methods of measuring DXP pathway polypeptideactivity are described in more detail in International Publication No.:WO 2010/148150

Exemplary DXS polypeptides include polypeptides, fragments ofpolypeptides, peptides, and fusions polypeptides that have at least oneactivity of a DXS polypeptide. Standard methods (such as those describedherein) can be used to determine whether a polypeptide has DXSpolypeptide activity by measuring the ability of the polypeptide toconvert pyruvate and D-glyceraldehyde-3-phosphate into1-deoxy-D-xylulose-5-phosphate in vitro, in a cell extract, or in vivo.Exemplary DXS polypeptides and nucleic acids and methods of measuringDXS activity are described in more detail in International PublicationNo. WO 2009/076676, U.S. patent application Ser. No. 12/335,071 (USPubl. No. 2009/0203102), WO 2010/003007, US Publ. No. 2010/0048964, WO2009/132220, and US Publ. No. 2010/0003716.

In particular, DXS polypeptides convert pyruvate and D-glyceraldehyde3-phosphate into 1-deoxy-d-xylulose 5-phosphate (DXP). Standard methodscan be used to determine whether a polypeptide has DXS polypeptideactivity by measuring the ability of the polypeptide to convert pyruvateand D-glyceraldehyde 3-phosphate in vitro, in a cell extract, or invivo.

DXR polypeptides convert 1-deoxy-d-xylulose 5-phosphate (DXP) into2-C-methyl-D-erythritol 4-phosphate (MEP). Standard methods can be usedto determine whether a polypeptide has DXR polypeptides activity bymeasuring the ability of the polypeptide to convert DXP in vitro, in acell extract, or in vivo.

MCT polypeptides convert 2-C-methyl-D-erythritol 4-phosphate (MEP) into4-(cytidine 5′-diphospho)-2-methyl-D-erythritol (CDP-ME). Standardmethods can be used to determine whether a polypeptide has MCTpolypeptides activity by measuring the ability of the polypeptide toconvert MEP in vitro, in a cell extract, or in vivo.

CMK polypeptides convert 4-(cytidine5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME) into2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-MEP).Standard methods can be used to determine whether a polypeptide has CMKpolypeptides activity by measuring the ability of the polypeptide toconvert CDP-ME in vitro, in a cell extract, or in vivo.

MCS polypeptides convert 2-phospho-4-(cytidine5′-diphospho)-2-C-methyl-D-erythritol (CDP-MEP) into2-C-methyl-D-erythritol 2, 4-cyclodiphosphate (ME-CPP or cMEPP).Standard methods can be used to determine whether a polypeptide has MCSpolypeptides activity by measuring the ability of the polypeptide toconvert CDP-MEP in vitro, in a cell extract, or in vivo.

HDS polypeptides convert 2-C-methyl-D-erythritol 2, 4-cyclodiphosphateinto (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP or HDMAPP).Standard methods can be used to determine whether a polypeptide has HDSpolypeptides activity by measuring the ability of the polypeptide toconvert ME-CPP in vitro, in a cell extract, or in vivo.

HDR polypeptides convert (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphateinto isopentenyl diphosphate (IPP) and dimethylallyl diphosphate(DMAPP). Standard methods can be used to determine whether a polypeptidehas HDR polypeptides activity by measuring the ability of thepolypeptide to convert HMBPP in vitro, in a cell extract, or in vivo.

Source Organisms for MVA Pathway, Isoprene Synthase, IDI, and DXPPathway Polypeptides

Isoprene synthase, IDI, DXP pathway, and/or MVA pathway nucleic acids(and their encoded polypeptides) can be obtained from any organism thatnaturally contains isoprene synthase, IDI, DXP pathway, and/or MVApathway nucleic acids. Isoprene is formed naturally by a variety oforganisms, such as bacteria, yeast, plants, and animals. Some organismscontain the MVA pathway for producing isoprene. Isoprene synthasenucleic acids can be obtained, e.g., from any organism that contains anisoprene synthase. MVA pathway nucleic acids can be obtained, e.g., fromany organism that contains the MVA pathway. IDI and DXP pathway nucleicacids can be obtained, e.g., from any organism that contains the IDI andDXP pathway.

The nucleic acid sequence of the isoprene synthase, DXP pathway, IDI,and/or MVA pathway nucleic acids can be isolated from a bacterium,fungus, plant, algae, or cyanobacterium. Exemplary source organismsinclude, for example, yeasts, such as species of Saccharomyces (e.g., S.cerevisiae), bacteria, such as species of Escherichia (e.g., E. coli),or species of Methanosarcina (e.g., Methanosarcina mazei), plants, suchas kudzu or poplar (e.g., Populus alba or Populus alba x tremulaCAC35696) or aspen (e.g., Populus tremuloides). Exemplary sources forisoprene synthases, IDI, and/or MVA pathway polypeptides which can beused are also described in International Patent Application PublicationNos. WO2009/076676, WO2010/003007, WO2009/132220, WO2010/031062,WO2010/031068, WO2010/031076, WO2010/013077, WO2010/031079,WO2010/148150, WO2010/078457, and WO2010/148256.

Recombinant Cells Capable of Increased Production of Isoprene

The recombinant cells described herein that have been engineered forincreased carbon flux to isoprene have the ability to produce isopreneat a concentration greater than that of the same cells that have notbeen engineered for increased carbon flux to isoprene. In one aspect,the recombinant cells (such as bacterial, fungal, or algal cells)described herein comprising one or more acetylating proteins, whereinsaid proteins are engineered such that their expression and/or activityis modulated, have the ability to produce isoprene at a concentrationgreater than that of the same cells lacking one or more acetylatingproteins, wherein said proteins are engineered such that theirexpression and/or activity is modulated. The acetylating proteins can beacetyltransferases (such as, but not limited to, YfiQ) and/ordeacetylases (such as, but not limited to CobB). In some embodiments,the activity of the YfiQ polypeptide is modulated by decreasing,attenuating, or deleting the expression of the gene encoding the YfiQpolypeptide (such as, but not limited to, deletion of an endogenous yfiQgene). In other embodiments, the activity of the CobB polypeptide ismodulated by increasing the expression of the gene encoding the CobBprotein (such as, but not limited to, increasing the expression of anendogenous cobB gene or heterologous expression of a nucleic acidencoding cobB). In other embodiments, culturing these cells in asuitable media provides for improved production of isoprene compared toa cell producing isoprene that does not comprise one or more acetylatingproteins wherein said proteins are engineered such that their expressionand/or activity is modulated. In some aspects, the cells furthercomprise one or more heterologous nucleic acids encoding an isoprenesynthase polypeptide. In certain aspects, these cells can furthercomprise one or more copies of a heterologous nucleic acid encodingpolypeptides of the entire MVA pathway, one or more heterologous nucleicacids encoding a phosphoketolase polypeptide, and/or one or moreheterologous nucleic acids encoding an isoprene synthase polypeptide.The one or more heterologous nucleic acids can be integrated into thehost cell's chromosome, in any aspect of the cells disclosed herein. Inother aspects, improved production of isoprene is characterized by oneor more of an increase in isoprene specific productivity, an increase inisoprene titer, an increase in isoprene yield, an increase in cellviability, and/or a decrease in acetate production.

In one exemplary embodiment, the cells disclosed herein can produce atleast 5% greater amounts of isoprene compared to isoprene-producingcells that have not been engineered to increase carbon flux to isoprene.In other aspects, the cells (such as bacterial, fungal, or algal cells)can produce at least 5% greater amounts of isoprene compared toisoprene-producing cells (such as bacterial, fungal, or algal cells)that do not comprise one or more acetylating proteins wherein saidproteins are engineered such that their expression and/or activity ismodulated. Alternatively, the cells (such as bacterial, fungal, or algalcells) can produce greater than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, or 15% of isoprene, inclusive, as well asany numerical value in between these numbers.

In one aspect of the invention, there are provided cells that have beenengineered for increased carbon flux to isoprene wherein the cellscomprise one or more acetylating proteins, wherein said proteins areengineered such that their expression and/or activity is modulated, oneor more heterologous nucleic acids encoding polypeptides of the entireMVA pathway, one or more heterologous nucleic acids encoding aphosphoketolase polypeptide, and one or more heterologous nucleic acidsencoding an isoprene synthase polypeptide. In some aspects, the cellscan further comprise one or more heterologous nucleic acids encoding aDXP pathway polypeptide(s). The cells can further comprise one or moreheterologous nucleic acids encoding an IDI polypeptide. The one or moreheterologous nucleic acids can be operably linked to constitutivepromoters, can be operably linked to inducible promoters, or can beoperably linked to a combination of inducible and constitutivepromoters. The one or more heterologous nucleic acids can additionallybe operably linked strong promoters, weak promoters, and/or mediumpromoters. One or more of the heterologous nucleic acids can beintegrated into a genome of the host cells or can be stably expressed inthe cells. The one or more heterologous nucleic acids can additionallybe on a vector.

The production of isoprene by cells that have been engineered forincreased carbon flux to isoprene according to any of the compositionsor methods described herein can be enhanced (e.g., enhanced by theexpression of one or more acetylating proteins, wherein said proteinsare engineered such that their expression and/or activity is modulated).In other aspects, the production of isoprene by the cells according toany of the compositions or methods described herein can beenhanced/increased/improved (e.g., enhanced by the expression of one ormore acetylating proteins, wherein said proteins are engineered suchthat their expression and/or activity is modulated, one or moreheterologous nucleic acids encoding an isoprene synthase polypeptide,polypeptides of the entire MVA pathway, a DXP pathway polypeptide(s),and/or an IDI polypeptide). As used herein,“enhanced”/“improved”/“increased” isoprene production refers to anincreased cell productivity index (CPI) for isoprene, an increased titerof isoprene, an increased mass yield of isoprene, increase in thecumulative isoprene yield, an increase in late fermentation isopreneproduction, an increase in cell viability, a decrease in acetateproduction, and/or an increased specific productivity of isoprene by thecells described by any of the compositions and methods described hereincompared to cells which do not express one or more acetylating proteins,wherein said proteins are engineered such that their expression and/oractivity is modulated, and which have not been engineered for increasedcarbon flux to isoprene production. The production of isoprene can beenhanced by about 5% to about 1,000,000 folds. The production ofisoprene can be enhanced by about 10% to about 1,000,000 folds (e.g.,about 1 to about 500,000 folds, about 1 to about 50,000 folds, about 1to about 5,000 folds, about 1 to about 1,000 folds, about 1 to about 500folds, about 1 to about 100 folds, about 1 to about 50 folds, about 5 toabout 100,000 folds, about 5 to about 10,000 folds, about 5 to about1,000 folds, about 5 to about 500 folds, about 5 to about 100 folds,about 10 to about 50,000 folds, about 50 to about 10,000 folds, about100 to about 5,000 folds, about 200 to about 1,000 folds, about 50 toabout 500 folds, or about 50 to about 200 folds) compared to theproduction of isoprene by cells that do not express one or moreacetylating proteins, wherein said proteins are engineered such thattheir expression and/or activity is modulated.

The production of isoprene can also be enhanced by at least about any of10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 5 folds,10 folds, 20 folds, 50 folds, 100 folds, 200 folds, 500 folds, 1000folds, 2000 folds, 5000 folds, 10,000 folds, 20,000 folds, 50,000 folds,100,000 folds, 200,000 folds, 500,000 folds, or 1,000,000 folds.

Methods of Using the Recombinant Cells to Produce Isoprene

Also provided herein are methods of producing isoprene comprisingculturing any of the recombinant microorganisms that have beenengineered for increased carbon flux to isoprene as described herein. Inone aspect, isoprene can be produced by culturing recombinant cells(such as bacterial, fungal, or algal cells) comprising one or morenucleic acids encoding one or more acetylating proteins, wherein saidcells have been engineered such that the expression of the nucleic acidsand/or activity of the acetylating protein(s) is modulated, one or moreheterologous nucleic acids encoding polypeptides of the entire MVApathway, and an isoprene synthase polypeptide. In certain embodiments,the recombinant cells can further comprise one or more nucleic acidsencoding a phosphoketolase polypeptide. The isoprene can be producedfrom any of the cells described herein and according to any of themethods described herein. Any of the cells can be used for the purposeof producing isoprene from carbohydrates, such as, but not limited to,glucose or from other carbon sources, such as, but not limited to,acetate.

Thus, also provided herein are methods of producing isoprene comprising(a) culturing cells which comprise one or more acetylating proteinswherein the cells have been engineered such that the expression and/oractivity of the acetylating proteins is modulated; and (b) producingisoprene. In other aspects, provided herein are methods of producingisoprene comprising (a) culturing cells (such as bacterial, fungal, oralgal cells) comprising one or more acetylating proteins, wherein saidproteins are engineered such that their activity is modulated, in asuitable media for producing isoprene and (b) producing isoprene. Thecells can comprise one or more nucleic acid molecules encodingpolypeptides of the entire MVA pathway as described above, one or morenucleic acid molecules encoding a phosphoketolase polypeptide, and anyof the isoprene synthase polypeptide(s) described above. In someaspects, the cells (such as bacterial, fungal, or algal cells) can beany of the cells described herein. Any of the isoprene synthases orvariants thereof described herein, any of the microorganism (e.g.,bacterial) or plant strains described herein, any of the promotersdescribed herein, and/or any of the vectors described herein can also beused to produce isoprene using any of the energy sources (e.g. glucoseor any other six carbon sugar) described herein. In some aspects, themethod of producing isoprene further comprises a step of recovering theisoprene.

In some aspects, the amount of isoprene produced is measured at the peakabsolute productivity time point. In some aspects, the peak absoluteproductivity for the cells is about any of the amounts of isoprenedisclosed herein. In some aspects, the amount of isoprene produced ismeasured at the peak specific productivity time point. In some aspects,the peak specific productivity for the cells is about any of the amountsof isoprene per cell disclosed herein. In some aspects, the cumulative,total amount of isoprene produced is measured. In some aspects, thecumulative total productivity for the cells is about any of the amountsof isoprene disclosed herein.

In some aspects, any of the cells described herein that have beenengineered such that the expression and/or activity of the acetylatingproteins are modulated (for examples the cells in culture) produceisoprene at greater than about any of or about any of 1, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250,1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or more nmole ofisoprene/gram of cells for the wet weight of the cells/hour(nmole/g_(wcm)/hr). In some aspects, the amount of isoprene is betweenabout 2 to about 5,000 nmole/g_(wcm)/hr, such as between about 2 toabout 100 nmole/g_(wcm)/hr, about 100 to about 500 nmole/g_(wcm)/hr,about 150 to about 500 nmole/g_(wcm)/hr, about 500 to about 1,000nmole/g_(wcm)/hr, about 1,000 to about 2,000 nmole/g_(wcm)/hr, or about2,000 to about 5,000 nmole/g_(wcm)/hr. In some aspects, the amount ofisoprene is between about 20 to about 5,000 nmole/g_(wcm)/hr, about 100to about 5,000 nmole/g_(wcm)/hr, about 200 to about 2,000nmole/g_(wcm)/hr, about 200 to about 1,000 nmole/g_(wcm)/hr, about 300to about 1,000 nmole/g_(wcm)/hr, or about 400 to about 1,000nmole/g_(wcm)/hr.

In some aspects, the cells that have been engineered such that theexpression and/or activity of the acetylating proteins are modulatedproduce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750,2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng ofisoprene/gram of cells for the wet weight of the cells/hr(ng/g_(wcm)/h). In some aspects, the amount of isoprene is between about2 to about 5,000 ng/g_(wcm)/h, such as between about 2 to about 100ng/g_(wcm)/h, about 100 to about 500 ng/g_(wcm)/h, about 500 to about1,000 ng/g_(wcm)/h, about 1,000 to about 2,000 ng/g_(wcm)/h, or about2,000 to about 5,000 ng/g_(wcm)/h. In some aspects, the amount ofisoprene is between about 20 to about 5,000 ng/g_(wcm)/h, about 100 toabout 5,000 ng/g_(wcm)/h, about 200 to about 2,000 ng/g_(wcm)/h, about200 to about 1,000 ng/g_(wcm)/h, about 300 to about 1,000 ng/g_(wcm)/h,or about 400 to about 1,000 ng/g_(wcm)/h.

In some aspects, the cells that have been engineered such that theexpression and/or activity of the acetylating proteins are modulatedproduce a cumulative titer (total amount) of isoprene at greater thanabout any of or about any of 1, 10, 25, 50, 100, 150, 200, 250, 300,400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500,3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of isoprene/Lof broth (mg/L_(broth), wherein the volume of broth includes the volumeof the cells and the cell medium). In some aspects, the amount ofisoprene is between about 2 to about 5,000 mg/L_(broth), such as betweenabout 2 to about 100 mg/L_(broth), about 100 to about 500 mg/L_(broth),about 500 to about 1,000 mg/L_(broth), about 1,000 to about 2,000mg/L_(broth), or about 2,000 to about 5,000 mg/L_(broth). In someaspects, the amount of isoprene is between about 20 to about 5,000mg/L_(broth), about 100 to about 5,000 mg/L_(broth), about 200 to about2,000 mg/L_(broth), about 200 to about 1,000 mg/L_(broth), about 300 toabout 1,000 mg/L_(broth), or about 400 to about 1,000 mg/L_(broth).

In some aspects, the cells that have been engineered such that theexpression and/or activity of the acetylating proteins are modulatedcomprises at least about 1, 2, 5, 10, 15, 20, or 25% by volume of thefermentation offgas. In some aspects, the isoprene comprises betweenabout 1 to about 25% by volume of the offgas, such as between about 5 toabout 15%, about 15 to about 25%, about 10 to about 20%, or about 1 toabout 10%.

The production of isoprene by recombinant cells described herein whichhave been engineered such that the expression and/or activity of theacetylating proteins in these cells is modulated can be enhanced byabout 5% to about 1,000,000 folds. The production of isoprene can beenhanced by about 10% to about 1,000,000 folds (e.g., about 50% to about1,000,000 folds, about 1 to about 500,000 folds, about 1 to about 50,000folds, about 1 to about 5,000 folds, about 1 to about 1,000 folds, about1 to about 500 folds, about 1 to about 100 folds, about 1 to about 50folds, about 5 to about 100,000 folds, about 5 to about 10,000 folds,about 5 to about 1,000 folds, about 5 to about 500 folds, about 5 toabout 100 folds, about 10 to about 50,000 folds, about 50 to about10,000 folds, about 100 to about 5,000 folds, about 200 to about 1,000folds, about 50 to about 500 folds, or about 50 to about 200 folds)compared to the production of isoprene by the cells that express wildtype levels of one or more acetylating proteins, one or moreheterologous nucleic acids encoding an isoprene synthase polypeptide,one or more heterologous nucleic acids encoding polypeptide of theentire MVA pathway, a DXP pathway polypeptide(s), and/or an IDIpolypeptide and which have not been engineered for increased carbon fluxto isoprene production.

The production of isoprene can also be enhanced by at least about any of10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1 fold, 2 folds, 5 folds,10 folds, 20 folds, 50 folds, 100 folds, 200 folds, 500 folds, 1000folds, 2000 folds, 5000 folds, 10,000 folds, 20,000 folds, 50,000 folds,100,000 folds, 200,000 folds, 500,000 folds, or 1,000,000 folds comparedto the production of isoprene by naturally-occurring cells or by cellsthat do not express one or more acetylating proteins, wherein saidproteins are engineered such that their activity is modulated, one ormore heterologous nucleic acids encoding an isoprene synthasepolypeptide, one or more heterologous nucleic acids encoding polypeptideof the entire MVA pathway, a DXP pathway polypeptide(s), and/or an IDIpolypeptide and which have not been engineered for increased carbon fluxto isoprene production.

Recombinant Cells Capable of Production of Isoprenoid Precursors and/orIsoprenoids

Isoprenoids can be produced in many organisms from the synthesis of theisoprenoid precursor molecules which are the end products of the MVApathway. As stated above, isoprenoids represent an important class ofcompounds and include, for example, food and feed supplements, flavorand odor compounds, and anticancer, antimalarial, antifungal, andantibacterial compounds.

As a class of molecules, isoprenoids are classified based on the numberof isoprene units comprised in the compound. Monoterpenes comprise tencarbons or two isoprene units, sesquiterpenes comprise 15 carbons orthree isoprene units, diterpenes comprise 20 carbons or four isopreneunits, sesterterpenes comprise 25 carbons or five isoprene units, and soforth. Steroids (generally comprising about 27 carbons) are the productsof cleaved or rearranged isoprenoids.

Isoprenoids can be produced from the isoprenoid precursor molecules IPPand DMAPP. These diverse compounds are derived from these rather simpleuniversal precursors and are synthesized by groups of conservedpolyprenyl pyrophosphate synthases (Hsieh et al., Plant Physiol. 2011March; 155(3):1079-90). The various chain lengths of these linear prenylpyrophosphates, reflecting their distinctive physiological functions, ingeneral are determined by the highly developed active sites ofpolyprenyl pyrophosphate synthases via condensation reactions of allylicsubstrates (dimethylallyl diphosphate (C₅-DMAPP), geranyl pyrophosphate(C₁₀-GPP), farnesyl pyrophosphate (C₁₅-FPP), geranylgeranylpyrophosphate (C₂₀-GGPP)) with corresponding number of isopentenylpyrophosphates (C₁₅-IPP) (Hsieh et al., Plant Physiol. 2011 March;155(3):1079-90). Examples of polyprenyl pyrophosphate synthases include,but are not limited to, farnesyl pyrophosphate (FPP) synthase (e.g.famesene synthase codon-optimized for E. coli (SEQ ID NO:26) oramorphadiene synthase codon-optimized for E. coli (SEQ ID NO:25));geranyl pyrophosphate synthase; or geranylgeranyl pyrophosphatesynthase.

Production of isoprenoid precursors and/or isoprenoids can be made byusing any of the recombinant host cells described here where one or moreof the enzymatic pathways have been manipulated such that enzymeactivity is modulated to increase carbon flow towards isoprenoidproduction. In addition, these cells can express one or more nucleicacids encoding one or more acetylating proteins, wherein said cells havebeen engineered such that the expression of the nucleic acids and/oractivity of the acetylating protein(s) is modulated, one or moreheterologous nucleic acids encoding polypeptides of the entire MVApathway, and/or one or more heterologous nucleic acids encoding aphosphoketolase polypeptide, and/or one or more heterologous nucleicacids encoding an isoprene synthase polypeptide. In some aspects, thesecells further comprise one or more heterologous nucleic acids encodingIDI and/or the DXP pathway polypeptides, as described above, and/or aheterologous nucleic acid encoding a polyprenyl pyrophosphate synthasepolypeptide. Without being bound to theory, it is thought thatincreasing the cellular production of acetyl-CoA or mevalonate in cellsby any of the compositions and methods described above will similarlyresult in the production of higher amounts of isoprenoid precursormolecules and/or isoprenoids. Increasing the molar yield of acetyl-CoAand/or mevalonate production from glucose translates into higher molaryields of isoprenoid precursor molecules and/or isoprenoids, includingisoprene, produced from glucose when combined with appropriate enzymaticactivity levels of mevalonate kinase, phosphomevalonate kinase,diphosphomevalonate decarboxylase, isopentenyl diphosphate isomerase andother appropriate enzymes for isoprene and isoprenoid production.

Types of Isoprenoids

The cells of the present invention that have been engineered forincreased carbon flux to mevalonate are capable of increased productionof isoprenoids and the isoprenoid precursor molecules DMAPP and IPP.Examples of isoprenoids include, without limitation, hemiterpenes,monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids,triterpenoids, tetraterpenoids, and higher polyterpenoids. In someaspects, the hemiterpenoid is prenol (i.e., 3-methyl-2-buten-1-ol),isoprenol (i.e., 3-methyl-3-buten-1-ol), 2-methyl-3-buten-2-ol, orisovaleric acid. In some aspects, the monoterpenoid can be, withoutlimitation, geranyl pyrophosphate, eucalyptol, limonene, or pinene. Insome aspects, the sesquiterpenoid is farnesyl pyrophosphate,artemisinin, or bisabolol. In some aspects, the diterpenoid can be,without limitation, geranylgeranyl pyrophosphate, retinol, retinal,phytol, taxol, forskolin, or aphidicolin. In some aspects, thetriterpenoid can be, without limitation, squalene or lanosterol. Theisoprenoid can also be selected from the group consisting ofabietadiene, amorphadiene, carene, α-famesene, β-famesene, farnesol,geraniol, geranylgeraniol, linalool, limonene, myrcene, nerolidol,ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpindene andvalencene.

In some aspects, the tetraterpenoid is lycopene or carotene (acarotenoid). As used herein, the term “carotenoid” refers to a group ofnaturally-occurring organic pigments produced in the chloroplasts andchromoplasts of plants, of some other photosynthetic organisms, such asalgae, in some types of fungus, and in some bacteria. Carotenoidsinclude the oxygen-containing xanthophylls and the non-oxygen-containingcarotenes. In some aspects, the carotenoids are selected from the groupconsisting of xanthophylls and carotenes. In some aspects, thexanthophyll is lutein or zeaxanthin. In some aspects, the carotenoid isα-carotene, β-carotene, γ-carotene, β-cryptoxanthin or lycopene.

Heterologous Nucleic Acids Encoding Polyprenyl Pyrophosphate SynthasesPolypeptides

In some aspects of the invention, the cells that have been engineeredfor increased carbon flux to isoprenoids described in any of thecompositions or methods herein further comprise one or more nucleicacids encoding a lower mevalonate (MVA) pathway polypeptide(s), asdescribed above, as well as one or more nucleic acids encoding apolyprenyl pyrophosphate synthase polypeptide(s). The polyprenylpyrophosphate synthase polypeptide can be an endogenous polypeptide. Theendogenous nucleic acid encoding a polyprenyl pyrophosphate synthasepolypeptide can be operably linked to a constitutive promoter or cansimilarly be operably linked to an inducible promoter. The endogenousnucleic acid encoding a polyprenyl pyrophosphate synthase polypeptidecan additionally be operably linked to a strong promoter. Alternatively,the endogenous nucleic acid encoding a polyprenyl pyrophosphate synthasepolypeptide can be operably linked to a weak promoter. In particular,the cells can be engineered to over-express the endogenous polyprenylpyrophosphate synthase polypeptide relative to wild-type cells.

In some aspects, the polyprenyl pyrophosphate synthase polypeptide is aheterologous polypeptide. The cells of the present invention cancomprise more than one copy of a heterologous nucleic acid encoding apolyprenyl pyrophosphate synthase polypeptide. In some aspects, theheterologous nucleic acid encoding a polyprenyl pyrophosphate synthasepolypeptide is operably linked to a constitutive promoter. In someaspects, the heterologous nucleic acid encoding a polyprenylpyrophosphate synthase polypeptide is operably linked to an induciblepromoter. In some aspects, the heterologous nucleic acid encoding apolyprenyl pyrophosphate synthase polypeptide is operably linked to astrong promoter. In some aspects, the heterologous nucleic acid encodinga polyprenyl pyrophosphate synthase polypeptide is operably linked to aweak promoter.

The nucleic acids encoding a polyprenyl pyrophosphate synthasepolypeptide(s) can be integrated into a genome of the host cells or canbe stably expressed in the cells. The nucleic acids encoding apolyprenyl pyrophosphate synthase polypeptide(s) can additionally be ona vector.

Exemplary polyprenyl pyrophosphate synthase nucleic acids includenucleic acids that encode a polypeptide, fragment of a polypeptide,peptide, or fusion polypeptide that has at least one activity of apolyprenyl pyrophosphate synthase. Polyprenyl pyrophosphate synthasepolypeptides convert isoprenoid precursor molecules into more complexisoprenoid compounds. Exemplary polyprenyl pyrophosphate synthasepolypeptides include polypeptides, fragments of polypeptides, peptides,and fusions polypeptides that have at least one activity of an isoprenesynthase polypeptide. Exemplary polyprenyl pyrophosphate synthasepolypeptides and nucleic acids include naturally-occurring polypeptidesand nucleic acids from any of the source organisms described herein. Inaddition, variants of polyprenyl pyrophosphate synthase can possessimproved activity such as improved enzymatic activity. In some aspects,a polyprenyl pyrophosphate synthase variant has other improvedproperties, such as improved stability (e.g., thermo-stability), and/orimproved solubility. Exemplary polyprenyl pyrophosphate synthase nucleicacids can include nucleic acids which encode polyprenyl pyrophosphatesynthase polypeptides such as, without limitation, geranyl diphosphate(GPP) synthase, farnesyl pyrophosphate (FPP) synthase, andgeranylgeranyl pyrophosphate (GGPP) synthase, or any other knownpolyprenyl pyrophosphate synthase polypeptide.

In some aspects of the invention, the cells that have been engineeredfor increased carbon flux to isoprenoids described in any of thecompositions or methods herein further comprise one or more nucleicacids encoding a farnesyl pyrophosphate (FPP) synthase. The FPP synthasepolypeptide can be an endogenous polypeptide encoded by an endogenousgene. In some aspects, the FPP synthase polypeptide is encoded by anendogenous ispA gene in E. coli (e.g. SEQ ID NO:23). The endogenousnucleic acid encoding an FPP synthase polypeptide can be operably linkedto a constitutive promoter or can similarly be operably linked to aninducible promoter. The endogenous nucleic acid encoding an FPP synthasepolypeptide can additionally be operably linked to a strong promoter. Inparticular, the cells can be engineered to over-express the endogenousFPP synthase polypeptide relative to wild-type cells.

In some aspects, the FPP synthase polypeptide is a heterologouspolypeptide. The cells of the present invention can comprise more thanone copy of a heterologous nucleic acid encoding a FPP synthasepolypeptide. In some aspects, the heterologous nucleic acid encoding aFPP synthase polypeptide is operably linked to a constitutive promoter.In some aspects, the heterologous nucleic acid encoding a FPP synthasepolypeptide is operably linked to an inducible promoter. In someaspects, the heterologous nucleic acid encoding a polyprenylpyrophosphate synthase polypeptide is operably linked to a strongpromoter.

The nucleic acids encoding an FPP synthase polypeptide can be integratedinto a genome of the host cells or can be stably expressed in the cells.The nucleic acids encoding an FPP synthase can additionally be on avector.

Standard methods can be used to determine whether a polypeptide haspolyprenyl pyrophosphate synthase polypeptide activity by measuring theability of the polypeptide to convert IPP into higher order isoprenoidsin vitro, in a cell extract, or in vivo. These methods are well known inthe art and are described, for example, in U.S. Pat. No. 7,915,026;Hsieh et al., Plant Physiol. 2011 March; 155(3):1079-90; Danner et al.,Phytochemistry. 2011 Apr. 12 [Epub ahead of print]; Jones et al., J BiolChem. 2011 Mar. 24 [Epub ahead of print]; Keeling et al., BMC PlantBiol. 2011 Mar. 7; 11:43; Martin et al., BMC Plant Biol. 2010 Oct. 21;10:226; Kumeta & Ito, Plant Physiol. 2010 December; 154(4):1998-2007;and Köllner & Boland, J Org Chem. 2010 Aug. 20; 75(16):5590-600.

Recombinant Cells Capable of Increased Production of IsoprenoidPrecursors and/or Isoprenoids

The recombinant microorganisms (e.g., recombinant bacterial, fungal, oralgal cells) described herein have the ability to produce isoprenoidprecursors and/or isoprenoids at an amount and/or concentration greaterthan that of the same cells without any manipulation to the variousenzymatic pathways described herein. In addition, the cells describedherein have the ability to produce isoprenoid precursors and/orisoprenoids at an amount and/or concentration greater than that of thesame cells that have not been engineered for increased carbon flux toisoprenoids and which lack one or more nucleic acids encoding one ormore acetylating proteins, wherein the cells have been engineered suchthat the expression of the nucleic acids and/or activity of theacetylating protein(s) is modulated, and one or more heterologousnucleic acids encoding a polyprenyl pyrophosphate synthase polypeptide.The acetylating proteins can be acetyltransferases (such as, but notlimited to, YfiQ) and/or deacetylases (such as, but not limited toCobB). In some embodiments, the activity of the YfiQ polypeptide ismodulated by decreasing, attenuating, or deleting the expression of thegene encoding the YfiQ polypeptide (such as, but not limited to,deletion of an endogenous yfiQ gene). In other embodiments, the activityof the CobB polypeptide is modulated by increasing the activity of theCobB protein (such as, but not limited to, increasing the expression ofan endogenous cobB gene or heterologous expression of a nucleic acidencoding cobB). In other embodiments, culturing these cells in asuitable media provides for improved production of isoprenoid precursorsand/or isoprenoids compared to a cell producing isoprenoid precursorsand/or isoprenoids that does not comprise one or more acetylatingproteins, wherein said proteins are engineered such that their activityis modulated. In certain aspects, these cells can further comprise oneor more copies of a heterologous nucleic acid encoding polypeptides ofthe entire MVA pathway and/or one or more heterologous nucleic acidsencoding a phosphoketolase polypeptide. The one or more heterologousnucleic acids can be integrated into the host cell's chromosome, in anyaspect of the cells disclosed herein. In other aspects, improvedproduction of isoprenoid precursors and/or isoprenoid is characterizedby one or more of an increase in isoprenoid precursor and/or isoprenoidspecific productivity, an increase in isoprenoid precursor and/orisoprenoid titer, an increase in isoprenoid precursor and/or isoprenoidyield, an increase in cell viability, and/or a decrease in acetateproduction.

In one aspect of the invention, there are provided cells that have beenengineered for increased carbon flux to isoprenoids and/or isoprenoidprecursors comprising one or more nucleic acids encoding one or moreacetylating proteins, wherein said cells have been engineered such thatthe expression of the nucleic acids and/or activity of the acetylatingprotein(s) is modulated, one or more heterologous nucleic acids encodingpolypeptides of the entire MVA pathway, and/or one or more heterologousnucleic acids encoding a phosphoketolase polypeptide, and/or one or moreheterologous nucleic acids encoding a DXP pathway polypeptide(s), and/orone or more heterologous nucleic acids encoding polyprenyl pyrophosphatesynthase. The cells can further comprise one or more heterologousnucleic acids encoding an IDI polypeptide. Additionally, the polyprenylpyrophosphate synthase polypeptide can be an FPP synthase polypeptide.The one or more heterologous nucleic acids can be operably linked toconstitutive promoters, can be operably linked to inducible promoters,or can be operably linked to a combination of inducible and constitutivepromoters. The one or more heterologous nucleic acids can additionallybe operably linked strong promoters, weak promoters, and/or mediumpromoters. One or more of the heterologous nucleic acids encoding one ormore acetylating proteins, wherein said proteins are engineered suchthat their activity is modulated, one or more heterologous nucleic acidsencoding polypeptides of the entire MVA pathway, one or moreheterologous nucleic acids encoding a phosphoketolase polypeptide, oneor more heterologous nucleic acids encoding a DXP pathwaypolypeptide(s), and/or one or more heterologous nucleic acids encodingpolyprenyl pyrophosphate synthase can be integrated into a genome of thehost cells or can be stably expressed in the cells. The one or moreheterologous nucleic acids can additionally be on a vector.

Provided herein are methods of using any of the cells that have beenengineered for increased carbon flux to isoprenoids and/or isoprenoidprecursor described above for enhanced, improved, or increasedisoprenoid precursor and/or isoprenoid production. As used herein,“enhanced”/“improved”/“increased” isoprenoid precursor and/or isoprenoidproduction refers to an increased cell productivity index (CPI) forisoprenoid precursor and/or isoprenoid production, an increased titer ofisoprenoid precursors and/or isoprenoids, an increased mass yield ofisoprenoid precursors and/or isoprenoids, an increase in isoprenoidprecursor and/or isoprenoid specific productivity, an increase in cellviability, and/or a decrease in acetate production by the cellsdescribed by any of the compositions and methods described hereincompared to cells which do not comprise one or more nucleic acidsencoding one or more acetylating proteins, wherein said cells have beenengineered such that the expression of the nucleic acids and/or activityof the acetylating protein(s) is modulated. The production of isoprenoidprecursors and/or isoprenoids can be enhanced by about 5% to about1,000,000 folds. The production of isoprenoid precursors and/orisoprenoids can be enhanced by about 10% to about 1,000,000 folds (e.g.,about 1 to about 500,000 folds, about 1 to about 50,000 folds, about 1to about 5,000 folds, about 1 to about 1,000 folds, about 1 to about 500folds, about 1 to about 100 folds, about 1 to about 50 folds, about 5 toabout 100,000 folds, about 5 to about 10,000 folds, about 5 to about1,000 folds, about 5 to about 500 folds, about 5 to about 100 folds,about 10 to about 50,000 folds, about 50 to about 10,000 folds, about100 to about 5,000 folds, about 200 to about 1,000 folds, about 50 toabout 500 folds, or about 50 to about 200 folds) compared to theproduction of isoprenoid and/or isoprenoid precursors by cells withoutthe expression of one or more acetylating proteins, wherein saidproteins are engineered such that their activity is modulated.

The production of isoprenoid precursors and/or isoprenoids can also beenhanced by at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 50 folds, 100folds, 200 folds, 500 folds, 1000 folds, 2000 folds, 5000 folds, 10,000folds, 20,000 folds, 50,000 folds, 100,000 folds, 200,000 folds, 500,000folds, or 1,000,000 folds compared to the production of isoprenoidprecursors and/or isoprenoids by naturally-occurring cells or by cellswithout the expression of one or more acetylating proteins, wherein saidproteins are engineered such that their activity is modulated and whichhave not been engineered for increased carbon flux to isoprenoids and/orisoprenoid precursor production.

Methods of Using the Recombinant Cells to Produce Isoprenoids and/orIsoprenoid Precursor Molecules

Also provided herein are methods of producing isoprenoid precursormolecules and/or isoprenoids comprising culturing recombinantmicroorganisms (e.g., recombinant bacterial, fungal, or algal cells)that have been engineered in various enzymatic pathways described hereinand/or comprising one or more nucleic acids encoding one or moreacetylating proteins, wherein said cells have been engineered such thatthe expression of the nucleic acids and/or activity of the acetylatingprotein(s) is modulated, one or more heterologous nucleic acids encodingpolypeptides of the entire MVA pathway, and a polyprenyl pyrophosphatesynthase polypeptide. In certain embodiments, the recombinant cellsfurther comprise one or more nucleic acids encoding a phosphoketolasepolypeptide. The isoprenoid precursor molecules and/or isoprenoids canbe produced from any of the cells described herein and according to anyof the methods described herein. Any of the cells can be used for thepurpose of producing isoprenoid precursor molecules and/or isoprenoidsfrom carbohydrates, such as, but not limited to, glucose or from othercarbon sources, such as, but not limited to, acetate.

Thus, provided herein are methods of making isoprenoid precursormolecules and/or isoprenoids comprising (a) culturing cells that havebeen engineered for increased carbon flux to isoprenoids and/orisoprenoid precursors; and (b) producing isoprenoid precursor moleculesand/or isoprenoids. In other aspects, provided herein are methods ofmaking isoprenoid precursor molecules and/or isoprenoids comprising (a)culturing cells (such as bacterial, fungal, or algal cells) comprisingone or more nucleic acids encoding one or more acetylating proteins,wherein said cells have been engineered such that the expression of thenucleic acids and/or activity of the acetylating protein(s) ismodulated, in a suitable media for producing isoprene and (b) producingisoprenoid precursor molecules and/or isoprenoids. The cells can furthercomprise one or more nucleic acid molecules encoding polypeptides of theentire MVA pathway as described above and/or one or more nucleic acidmolecules encoding a phosphoketolase polypeptide. In some aspects, thecells (such as bacterial, fungal, or algal cells) can be any of thecells described herein. In some aspects, the method of producingisoprenoid precursor molecules and/or isoprenoids further comprises astep of recovering the isoprenoid precursor molecules and/orisoprenoids.

In one exemplary embodiment, the instant methods for the production ofisoprenoid precursor molecules and/or isoprenoids can produce at least5% greater amounts of isoprenoid precursors and/or isoprenoids whencompared to isoprenoids and/or isoprenoid precursor-producing cells thathave not been engineered for increased carbon flux to isoprenoids and/orisoprenoid precursors and that do not comprise one or more nucleic acidsencoding one or more acetylating proteins, wherein said cells have beenengineered such that the expression of the nucleic acids and/or activityof the acetylating protein(s) is modulated. In other aspects, providedherein are methods for the production of isoprenoid precursor moleculesand/or isoprenoids that in one exemplary embodiment can produce at least5% greater amounts of isoprenoid precursors and/or isoprenoids whencompared to isoprenoids and/or isoprenoid precursor-producing cells(such as bacterial, fungal, or algal cells) that do not comprise one ormore nucleic acids encoding one or more acetylating proteins, whereinsaid cells have been engineered such that the expression of the nucleicacids and/or activity of the acetylating protein(s) is modulated, andwhich have not been engineered for increased carbon flux to isoprenoidsand/or isoprenoid precursor production. Alternatively, the cells (suchas bacterial, fungal, or algal cells) can produce greater than about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% ofisoprenoid precursors and/or isoprenoids, inclusive. In some aspects,the method of producing isoprenoid precursor molecules and/orisoprenoids further comprises a step of recovering the isoprenoidprecursor molecules and/or isoprenoids.

Provided herein are methods of using any of the cells that have beenengineered for increased carbon flux to isoprenoids and/or isoprenoidprecursors described above for enhanced isoprenoid and/or isoprenoidprecursor molecule production. As used herein, “enhanced” isoprenoidprecursor and/or isoprenoid production refers to an increased cellproductivity index (CPI) for isoprenoid precursor and/or isoprenoidproduction, an increased titer of isoprenoid precursors and/orisoprenoids, an increased mass yield of isoprenoid precursors and/orisoprenoids, an increased specific productivity of isoprenoid precursorsand/or isoprenoids, increased cumulative yield isoprenoid precursorsand/or isoprenoids, an increase in late fermentation of isoprenoidprecursors and/or isoprenoids, and increase in cell viability, and/or adecrease in acetate production by the cells described by any of thecompositions and methods described herein compared to cells which do notcomprise one or more nucleic acids encoding one or more acetylatingproteins, wherein said cells have been engineered such that theexpression of the nucleic acids and/or activity of the acetylatingprotein(s) is modulated. The production of isoprenoid precursormolecules and/or isoprenoids can be enhanced by about 10% to about1,000,000 folds (e.g., about 1 to about 500,000 folds, about 1 to about50,000 folds, about 1 to about 5,000 folds, about 1 to about 1,000folds, about 1 to about 500 folds, about 1 to about 100 folds, about 1to about 50 folds, about 5 to about 100,000 folds, about 5 to about10,000 folds, about 5 to about 1,000 folds, about 5 to about 500 folds,about 5 to about 100 folds, about 10 to about 50,000 folds, about 50 toabout 10,000 folds, about 100 to about 5,000 folds, about 200 to about1,000 folds, about 50 to about 500 folds, or about 50 to about 200folds) compared to the production of isoprenoid precursor moleculesand/or isoprenoids by cells that do not comprise one or more nucleicacids encoding one or more acetylating proteins, wherein said cells havebeen engineered such that the expression of the nucleic acids and/oractivity of the acetylating protein(s) is modulated. In one exemplaryembodiment, the production of isoprenoid precursor molecules and/orisoprenoids can be enhanced by about 5% to about 1,000,000 folds.

The production of isoprenoid precursor molecules and/or isoprenoids canalso be enhanced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 1 fold, 2 folds, 5 folds, 10 folds, 20 folds, 50 folds,100 folds, 200 folds, 500 folds, 1000 folds, 2000 folds, 5000 folds,10,000 folds, 20,000 folds, 50,000 folds, 100,000 folds, 200,000 folds,500,000 folds, or 1,000,000 folds compared to the production ofisoprenoid precursor molecules and/or isoprenoids by cells that do notcomprise one or more nucleic acids encoding one or more acetylatingproteins, wherein said cells have been engineered such that theexpression of the nucleic acids and/or activity of the acetylatingprotein(s) is modulated.

Modulation of Additional Enzymatic Pathways for the Improved Productionof Mevalonate, Isoprene, Isoprenoids, Isoprenoid Precursors, andAcetyl-CoA-Derived Products

In some aspects of the invention, the recombinant cells described in anyof the compositions or methods described herein further comprise one ormore nucleic acids encoding one or more proteins, where the cells havebeen modified such that the expression of the nucleic acids and/oractivity of the proteins is modulated. Such further modulation ofadditional genes involved in the utilization of carbon during cellularmetabolism or that are implicated with respect to the availableintracellular supply of acetyl-CoA may also be modulated to improveproduction of mevalonate, isoprene, isoprenoid precursors, and/orisoprenoids. These include, but are not limited to the modulations ofpathways involving phosphofructokinase, modulations of pathwaysinvolving phosphoketolase, modulations of the pentose phosphate pathwayenzymes, modulations of enzymes involved in acetate production, acetatecycling, and acetyl-CoA production, modulations of pathways involvingthe Entner-Doudoroff pathway, modulations of pathways Involving theOxidative Branch of the Pentose Phosphate Pathway, and the like.

Nucleic Acids Encoding Phosphoketolase Polypeptides

In some aspects of the invention, the recombinant cells described in anyof the compositions or methods described herein can further comprise oneor more nucleic acids encoding a phosphoketolase polypeptide or apolypeptide having phosphoketolase activity. In some aspects, thephosphoketolase polypeptide is a heterologous polypeptide. In someaspects, the heterologous nucleic acid encoding a phosphoketolasepolypeptide is operably linked to a constitutive promoter. In someaspects, the heterologous nucleic acid encoding a phosphoketolasepolypeptide is operably linked to an inducible promoter. In someaspects, the heterologous nucleic acid encoding a phosphoketolasepolypeptide is operably linked to a strong promoter. In some aspects,more than one heterologous nucleic acid encoding a phosphoketolasepolypeptide is used (e.g., 2, 3, 4, or more copies of a heterologousnucleic acid encoding a phosphoketolase polypeptide). In some aspects,the heterologous nucleic acid encoding a phosphoketolase polypeptide isoperably linked to a weak promoter. In a particular aspect, the cellsare engineered to overexpress the endogenous phosphoketolase polypeptiderelative to wild-type cells.

Phosphoketolase enzymes catalyze the conversion of xylulose 5-phosphateto glyceraldehyde 3-phosphate and acetyl phosphate and/or the conversionof fructose 6-phosphate to erythrose 4-phosphate and acetyl phosphate.In certain embodiments, the phosphoketolase enzyme is capable ofcatalyzing the conversion of xylulose 5-phosphate to glyceraldehyde3-phosphate and acetyl phosphate. In other embodiments, thephosphoketolase enzyme is capable of catalyzing the conversion offructose 6-phosphate to erythrose 4-phosphate and acetyl phosphate.Thus, without being bound by theory, the expression of phosphoketolaseas set forth herein can result in an increase in the amount of acetylphosphate produced from a carbohydrate source. This acetyl phosphate canbe converted into acetyl-CoA which can then be utilized by the enzymaticactivities of the MVA pathway to produces mevalonate, isoprenoidprecursor molecules, isoprene and/or isoprenoids. Thus the amount ofthese compounds produced from a carbohydrate substrate may be increased.Alternatively, production of Acetyl-P and AcCoA can be increased withoutthe increase being reflected in higher intracellular concentration. Incertain embodiments, intracellular acetyl-P or acetyl-CoA concentrationswill remain unchanged or even decrease, even though the phosphoketolasereaction is taking place.

Exemplary phosphoketolase nucleic acids include nucleic acids thatencode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of a phosphoketolasepolypeptide. Exemplary phosphoketolase polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

Standard methods can be used to determine whether a polypeptide hasphosphoketolase peptide activity by measuring the ability of the peptideto convert D-fructose 6-phosphate or D-xylulose 5-phosphate intoacetyl-P. Acetyl-P can then be converted into ferryl acetyl hydroxamate,which can be detected spectrophotometrically (Meile et al., J. Bact.183:2929-2936, 2001). Any polypeptide identified as havingphosphoketolase peptide activity as described herein is suitable for usein the present invention.

In other aspects, exemplary phosphoketolase nucleic acids include, forexample, a phosphoketolase isolated from Lactobacillus reuteri,Bifidobacterium longum, Ferrimonas balearica, Pedobactor saltans,Streptomyces griseus, Mycoplasma hominis, and/or Nocardiopsisdassonvillei. Additional examples of phosphoketolase enzymes which canbe used herein are described in U.S. Pat. No. 7,785,858, which isincorporated by reference herein.

Pathways Involving the Entner-Doudoroff Pathway

The Entner-Doudoroff (ED) pathway is an alternative to theEmden-Meyerhoff-Parnass (EMP-glycolysis) pathway. Some organisms, likeE. coli, harbor both the ED and EMP pathways, while others have only oneor the other. Bacillus subtilis has only the EMP pathway, whileZymomonas mobilis has only the ED pathway (Peekhaus and Conway. 1998. J.Bact. 180:3495-3502; Stulke and Hillen. 2000. Annu. Rev. Microbiol. 54,849-880; Dawes et al. 1966. Biochem. J. 98:795-803).

Phosphogluconate dehydratase (edd) removes one molecule of H₂O from6-phospho-D-gluconate to form 2-dehydro-3-deoxy-D-gluconate 6-phosphate,while 2-keto-3-deoxygluconate 6-phosphate aldolase (eda) catalyzes analdol cleavage (Egan et al. 1992. J. Bact. 174:4638-4646). The two genesare in an operon.

Metabolites that can be directed into the phosphoketolase pathway canalso be diverted into the ED pathway. To avoid metabolite loss to theED-pathway, phosphogluconate dehydratase gene (e.g., the endogenousphosphogluconate dehydratase gene) and/or a 2-keto-3-deoxygluconate6-phosphate aldolase gene (e.g., the endogenous 2-keto-3-deoxygluconate6-phosphate aldolase gene) activity is attenuated. One way of achievingattenuation is by deleting phosphogluconate dehydratase (edd) and/or2-keto-3-deoxygluconate 6-phosphate aldolase (eda). This can beaccomplished by replacing one or both genes with a chloramphenicol orkanamycin cassette followed by looping out of the cassette. Withoutthese enzymatic activities, more carbon can flux through thephosphoketolase enzyme, thus increasing the yield of mevalonate,isoprene, isoprenoid precursor molecules, isoprenoids, and/or acetyl-CoAderived products.

The activity of phosphogluconate dehydratase (edd) and/or2-keto-3-deoxygluconate 6-phosphate aldolase (eda) can also be decreasedby other molecular manipulations of the enzymes. The decrease of enzymeactivity can be any amount of reduction of specific activity or totalactivity as compared to when no manipulation has been effectuated. Insome instances, the decrease of enzyme activity is decreased by at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99%.

In some cases, attenuating the activity of the endogenousphosphogluconate dehydratase gene and/or the endogenous2-keto-3-deoxygluconate 6-phosphate aldolase gene results in more carbonflux into the mevalonate dependent biosynthetic pathway in comparison tocells that do not have attenuated endogenous phosphogluconatedehydratase gene and/or endogenous acetate kinase2-keto-3-deoxygluconate6-phosphate aldolase gene expression.

Pathways Involving the Oxidative Branch of the Pentose Phosphate Pathway

E. coli uses the pentose phosphate pathway to break down hexoses andpentoses and to provide cells with intermediates for various anabolicpathways. It is also a major producer of NADPH. The pentose phosphatepathway is composed from an oxidative branch (with enzymes like glucose6-phosphate 1-dehydrogenase (zwf), 6-phosphogluconolactonase (pgl) or6-phosphogluconate dehydrogenase (gnd)) and a non-oxidative branch (withenzymes such as transketolase (tktA), transaldolase (talA or talB),ribulose-5-phosphate-epimerase and (or) ribose-5-phosphate epimerase)(Sprenger. 1995. Arch. Microbiol. 164:324-330).

In order to direct carbon towards the phosphoketolase enzyme, expressionand/or activity of proteins of the non-oxidative branch of the pentosephosphate pathway (transketolase, transaldolase,ribulose-5-phosphate-epimerase and (or) ribose-5-phosphate epimerase)can be modulated (e.g., increase enzyme activity) to allow more carbonto flux towards fructose 6-phosphate and xylulose 5-phosphate, therebyincreasing the eventual production of mevalonate, isoprene, isoprenoidprecursor molecules, isoprenoids, and/or acetyl-CoA derived products.Increase of transketolase, transaldolase, ribulose-5-phosphate-epimeraseand (or) ribose-5-phosphate epimerase activity can be any amount ofincrease of specific activity or total activity as compared to when nomanipulation has been effectuated. In some instances, the enzymeactivity is increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some aspects,the activity of transketolase, transaldolase,ribulose-5-phosphate-epimerase and (or) ribose-5-phosphate epimerase ismodulated by increasing the activity of an endogenous transketolase,transaldolase, ribulose-5-phosphate-epimerase and (or)ribose-5-phosphate epimerase. This can be accomplished by replacing theendogenous transketolase, transaldolase, ribulose-5-phosphate-epimeraseand (or) ribose-5-phosphate epimerase gene promoter with a syntheticconstitutively high expressing promoter. The genes encodingtransketolase, transaldolase, ribulose-5-phosphate-epimerase and (or)ribose-5-phosphate epimerase can also be cloned on a plasmid behind anappropriate promoter. The increase of the activity of transketolase,transaldolase, ribulose-5-phosphate-epimerase and (or)ribose-5-phosphate epimerase can result in more carbon flux into themevalonate dependent biosynthetic pathway in comparison to cells that donot have increased expression of transketolase, transaldolase,ribulose-5-phosphate-epimerase and (or) ribose-5-phosphate epimerase.

Pathways Involving Phosphofructokinase

Phosphofructokinase is a crucial enzyme of glycolysis which catalyzesthe phosphorylation of fructose 6-phosphate. E. coli has two isozymesencoded by pfkA and pfkB. Most of the phosphofructokinase activity inthe cell is due to pfkA (Kotlarz et al. 1975, Biochim. Biophys. Acta,381:257-268).

In order to direct carbon towards the phosphoketolase enzyme,phosphofructokinase expression can be modulated (e.g., decrease enzymeactivity) to allow more carbon to flux towards fructose 6-phosphate andxylulose 5-phosphate, thereby increasing the eventual production ofmevalonate, isoprene, isoprenoid precursor molecules, isoprenoids,and/or acetyl-CoA derived products. Decrease of phosphofructokinaseactivity can be any amount of reduction of specific activity or totalactivity as compared to when no manipulation has been effectuated. Insome instances, the decrease of enzyme activity is decreased by at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, or 100%. In some aspects, the activity of phosphofructokinaseis modulated by decreasing the activity of an endogenousphosphofructokinase. This can be accomplished by replacing theendogenous phosphofructokinase gene promoter with a syntheticconstitutively low expressing promoter. The gene encodingphosphofructokinase can also be deleted. The decrease of the activity ofphosphofructokinase can result in more carbon flux into the mevalonatedependent biosynthetic pathway in comparison to cells that do not havedecreased expression of phosphofructokinase.

Modulation of Genes Involved in Acetate Production, Acetate Cycling, andAcetyl-CoA Production

In order to produce useful industrial fermentation products,microorganisms (such as E. coli and yeasts) have been widely used ashost strains for high-cell-density fermentations. A substantial amountof glucose is added into the growth medium for high-density growth ofhost cells as well as for expression of heterologously expressedrecombinant genes, since glucose is a relatively inexpensive and readilyutilizable carbon and energy source. One major problem that can occurduring high-cell-density fermentation is the production of fermentativeacidic by-products, of which acetate is the most predominant, that canbe a major factor in the limitation of cellular growth and production(Han et al., Biotechnol. Bioeng., 39, 663 (1992); Luli et al., Appl.Environ. Microbiol., 56,1004 (1990)).

In the embodiments provided herein, acetate cycling proteins, acetateproduction proteins, or Acetyl-CoA production-related proteins include,but are not limited to Phosphotransacetylase (Pta), acetate kinase(AckA), AMP-forming acetyl-coenzyme A synthetase (Acs), and acetatetransporter/acetate pump/(actP). In any one of the embodiments describedherein, mutations to one or more genes encoding these proteins increaseor decrease/attenuate/delete expression, or changes to the activities ofthese proteins can be made either singly, or in combination, to furtherenhance the production of mevalonate, isoprene, isoprenoids, isoprenoidprecursors, and/or acetyl-CoA-derived products. Changes to theexpression and/or activity of these acetate cycling proteins can be madesingly or in combination with modulation of the activity of one or moreacetylating proteins (such as, but not limited to YfiQ and/or CobB, asdescribed above). In some embodiments, the activity of anacetyltransferase is modulated by deleting or attenuating the expressionof the acetyltransferase polypeptide or the acetyltransferase gene alongwith modulation of one or more acetate cycling genes/proteins. Inexemplary embodiments, the activity of YfiQ is modulated by deleting orattenuating the expression of the YfiQ polypeptide or the yfiQ gene. Inother embodiments, the activity of a deacetylase is modulated byincreasing the expression or activity of the deacetylase polypeptide orthe deacetylase gene along with modulation of one or more acetatecycling genes/proteins. In exemplary embodiments, the activity of CobBis modulated by increasing the expression or activity of the CobBpolypeptide or the cobB gene.

For glucose metabolism in microorganisms under aerobic conditions,carbon flow exceeding the capacity of the Kreb's cycle (the TCA cycle),is converted to acetic acid/acetate which is ultimately excreted outsidethe cell (Majewski & Domach, Biotechnol. Bioeng., 35, 732 (1990)). Theexcreted acetic acid/acetate can inhibit the growth of the host strainand the production of the desired fermentation product.

Phosphotransacetylase (Pta) (Shimizu et al. 1969. Biochim. Biophys. Acta191: 550-558) catalyzes the reversible conversion between acetyl-CoA andacetyl phosphate (referred to interchangeably herein as acetylphosphate,acetyl-phosphate, acetyl-P, or Ac-P), while acetate kinase (AckA)(Kakuda, H. et al. 1994. J. Biochem. 11:916-922) uses acetyl phosphateto form acetate. The genes encoding these proteins can be transcribed asan operon in E. coli. Together, they catalyze the dissimilation ofacetate, with the release of ATP. Thus, one of skill in the art canincrease the amount of available acetyl-CoA by modulating the activityof phosphotransacetylase gene (e.g., the endogenousphosphotransacetylase gene) and/or an acetate kinase gene (e.g., theendogenous acetate kinase gene). For example, such modulation can beachieved by increasing the expression of a phosphotransacetylase gene.Such modulation can also be achieved by increasing the expression of theacetate kinase gene. In a particular embodiment, the modulation can beachieved by altering the expression of both the phosphotransacetylaseand acetate kinase genes. The modulation can also be achieved bydecreasing, attenuating, or deleting the expression of aphosphotransacetylase gene and/or an acetate kinase gene. One way ofachieving attenuation is by deleting the phosphotransacetylase (pta)and/or acetate kinase (ackA) genes. This can be accomplished byreplacing one or both genes with a chloramphenicol cassette followed bylooping out of the cassette. Without being bound by theory, deletingthese genes could increase the yield of mevalonate, isoprene orisoprenoids by diverting more carbon into the mevalonate pathway andaway from production of acetate.

Alternatively, without being bound by theory, increasing the expressionor activity of ackA can increase the production of acetate to be used tosynthesize acetyl-CoA.

Alternatively, without being bound by theory, increasing the expressionor activity of pta can increase the production of acetyl-CoA.

Further, modulation of the expression of pta and/or an ackA gene can beperformed in combination with modulation of the activity of one or moreacetylating proteins (such as, but not limited to YfiQ and/or CobB, asdescribed above). In some embodiments, the activity of anacetyltransferase is modulated by deleting or attenuating the expressionof the acetyltransferase polypeptide or the acetyltransferase gene. Inexemplary embodiments, the activity of YfiQ is modulated by deleting orattenuating the expression of the YfiQ polypeptide or the yfiQ gene. Inother embodiments, the activity of a deacetylase is modulated byincreasing the expression or activity of the deacetylase polypeptide orthe deacetylase gene. In exemplary embodiments, the activity of CobB ismodulated by increasing the expression or activity of the CobBpolypeptide or the cobB gene.

Another protein involved in acetate production and acetate cycling inmicroorganisms is AMP-forming acetyl-coenzyme A synthetase (Acs), whichis a ubiquitous enzyme responsible for the conversion of acetate to thehigh energy intermediate acetyl-CoA, a keystone molecule of centralmetabolism (Cerezo et al., 2011, Molec. Microb., 82(5):1110-28). Withoutbeing bound to theory, cells engineered to increase the expression ofAcs could be expected to produce higher amounts of acetyl-CoA.Additional, Acs is a substrate for acetyltransferases and deacetylases.By way of example only, the deacetylase activity of CobB has beendemonstrated on Acs in vitro (Zhao et al., 2004, J Mol. Biol.,337:731-41). Without being bound to theory, since acetylation of Acs canresults in its enzymatic inactivation, cells engineered to decrease theamount of Acs acetylation could be expected to produce higher amounts ofacetyl-CoA. Further, modulation of the expression of the Acs gene can beperformed in combination with modulation of the activity of one or moreacetylating proteins (such as, but not limited to YfiQ and/or CobB, asdescribed above). In some embodiments, the activity of anacetyltransferase is modulated by deleting or attenuating the expressionof the acetyltransferase polypeptide or the acetyltransferase gene. Inexemplary embodiments, the activity of YfiQ is modulated by deleting orattenuating the expression of the YfiQ polypeptide or the yfiQ gene. Inother embodiments, the activity of a deacetylase is modulated byincreasing the expression or activity of the deacetylase polypeptide orthe deacetylase gene. In exemplary embodiments, the activity of CobB ismodulated by increasing the expression or activity of the CobBpolypeptide or the cobB gene.

Another protein involved in acetate production and acetate handling inmicroorganisms is the acetate transporter/acetate pump (actP). actPactivity can be decreased or attenuated to minimize transport of acetateacross the membrane. Without being bound to theory, it is believed thatif acetate production is coupled with transport across the membrane,this could result in energy loss due to decoupling of the protongradient. In some aspects, decreased activity of actP or lack of actPcan be used to improve production of mevalonate, isoprene, isoprenoidprecursors, and isoprenoids. A modified actP gene may be introducedusing chromosomal integration or extra-chromosomal vehicles, such asplasmids. In yet other aspects, actP may be deleted from the genome ofcells (for example, microorganisms, such as various E. coli strains)which express a actP to improve production of mevalonate and/orisoprene. In another aspect, a heterologous nucleic acid encoding a actPpolypeptide can be expressed in a cell which does not endogenouslyexpress actP. Further, modulation of the expression of the actP gene canbe performed in combination with modulation of the activity of one ormore acetylating proteins (such as, but not limited to YfiQ and/or CobB,as described above). In some embodiments, the activity of anacetyltransferase is modulated by deleting or attenuating the expressionof the acetyltransferase polypeptide or the acetyltransferase gene. Inexemplary embodiments, the activity of YfiQ is modulated by deleting orattenuating the expression of the YfiQ polypeptide or the yfiQ gene. Inother embodiments, the activity of a deacetylase is modulated byincreasing the expression or activity of the deacetylase polypeptide orthe deacetylase gene. In exemplary embodiments, the activity of CobB ismodulated by increasing the expression or activity of the CobBpolypeptide or the cobB gene.

In some aspects, deletion or attenuation of actP, ackA, and/or ptaresults in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%, inclusive, such as any values in between these percentages, higherpercent yield of isoprene in comparison to microorganisms that expressactP, ackA, and/or pta. In other aspects, deletion of actP, ackA, or ptaresults in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%, inclusive, including any values in between these percentages,higher instantaneous percent yield of isoprene in comparison tomicroorganisms that express actP, ackA, and/or pta. In other aspects,deletion of actP, ackA, and/or pta results in any of about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive, including anyvalues in between these percentages, higher cell productivity index forisoprene in comparison to microorganisms that express actP, ackA, and/orpta. In other aspects, deletion of actP, ackA, and/or pta results in anyof about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%,inclusive, including any values in between these percentages, highervolumetric productivity of isoprene in comparison to microorganisms thatexpress actP, ackA, and/or pta. In other aspects, deletion of actP,ackA, and/or pta results in any of about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100%, inclusive, including any values in between thesepercentages, higher peak specific productivity of isoprene in comparisonto microorganisms that express actP, ackA, and/or pta. In some aspectsthe deletion of actP, ackA, and/or pta results in peak specificproductivity being maintained for a longer period of time in comparisonto microorganisms that express actP, ackA, and/or pta.

In some aspects, the recombinant microorganism produces decreasedamounts of acetate in comparison to microorganisms that do not haveattenuated endogenous phosphotransacetylase gene and/or endogenousacetate kinase gene expression. Decrease in the amount of acetateproduced can be measured by routine assays known to one of skill in theart. The amount of acetate reduction is at least about 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% ascompared when no molecular manipulations are done.

The activity of actP, ackA, and/or pta can also be decreased by othermolecular manipulation of the enzymes. The decrease of enzyme activitycan be any amount of reduction of specific activity or total activity ascompared to when no manipulation has been effectuated. In someinstances, the decrease of enzyme activity is decreased by at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99%.

In some cases, attenuating the activity of the endogenousphosphotransacetylase gene and/or the endogenous acetate kinase generesults in more carbon flux into the mevalonate dependent biosyntheticpathway in comparison to microorganisms that do not have attenuatedendogenous phosphotransacetylase gene and/or endogenous acetate kinasegene expression.

In some embodiments, the activity of AMP-forming acetyl-coenzyme Asynthetase (Acs), phosphotransacetylase (pta) and/or acetate kinase(ackA) can be increased. The increase of these enzymes' activity can beany amount of increase of specific activity or total activity ascompared to when no manipulation has been effectuated. In someinstances, the increase of enzyme activity is increased by at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99%.

Citrate Synthase Pathway

Citrate synthase catalyzes the condensation of oxaloacetate andacetyl-CoA to form citrate, a metabolite of the Tricarboxylic acid (TCA)cycle (Ner, S. et al. 1983. Biochemistry, 22: 5243-5249; Bhayana, V. andDuckworth, H. 1984. Biochemistry 23: 2900-2905). In E. coli, thisenzyme, encoded by gltA, behaves like a trimer of dimeric subunits. Thehexameric form allows the enzyme to be allosterically regulated by NADH.This enzyme has been widely studied (Wiegand, G., and Remington, S.1986. Annual Rev. Biophysics Biophys. Chem. 15: 97-117; Duckworth et al.1987. Biochem Soc Symp. 54:83-92; Stockell, D. et al. 2003. J Biol.Chem. 278: 35435-43; Maurus, R. et al. 2003. Biochemistry.42:5555-5565). To avoid allosteric inhibition by NADH, replacement by orsupplementation with the Bacillus subtilis NADH-insensitive citratesynthase has been considered (Underwood et al. 2002. Appl. Environ.Microbiol. 68:1071-1081; Sanchez et al. 2005. Met. Eng. 7:229-239).

The reaction catalyzed by citrate synthase directly competes with thethiolase catalyzing the first step of the mevalonate pathway, as theyboth have acetyl-CoA as a substrate (Hedl et al. 2002. J. Bact.184:2116-2122). Therefore, one of skill in the art can modulate citratesynthase expression (e.g., decrease enzyme activity) to allow morecarbon to flux into the mevalonate pathway, thereby increasing theeventual production of mevalonate, isoprene and isoprenoids. Decreasedcitrate synthase activity can be any amount of reduction of specificactivity or total activity as compared to when no manipulation has beeneffectuated. In some instances, the decrease of enzyme activity isdecreased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the activity ofcitrate synthase is modulated by decreasing the activity of anendogenous citrate synthase gene. This can be accomplished bychromosomal replacement of an endogenous citrate synthase gene with atransgene encoding an NADH-insensitive citrate synthase or by using atransgene encoding an NADH-insensitive citrate synthase that is derivedfrom Bacillus subtilis. The activity of citrate synthase can also bemodulated (e.g., decreased) by replacing the endogenous citrate synthasegene promoter with a synthetic constitutively low expressing promoter.The decrease of the activity of citrate synthase can result in morecarbon flux into the mevalonate dependent biosynthetic pathway incomparison to microorganisms that do not have decreased expression ofcitrate synthase.

Pathways Involving Lactate Dehydrogenase

In E. coli, D-Lactate is produced from pyruvate through the enzymelactate dehydrogenase (LdhA) (Bunch, P. et al. 1997. Microbiol.143:187-195). Production of lactate is accompanied with oxidation ofNADH, hence lactate is produced when oxygen is limited and cannotaccommodate all the reducing equivalents. Thus, production of lactatecould be a source for carbon consumption. As such, to improve carbonflow through to mevalonate production (and isoprene, isoprenoidprecursor and isoprenoids production, if desired), one of skill in theart can modulate the activity of lactate dehydrogenase, such as bydecreasing the activity of the enzyme.

Accordingly, in one aspect, the activity of lactate dehydrogenase can bemodulated by attenuating the activity of an endogenous lactatedehydrogenase gene. Such attenuation can be achieved by deletion of theendogenous lactate dehydrogenase gene. Other ways of attenuating theactivity of lactate dehydrogenase gene known to one of skill in the artmay also be used.

By manipulating the pathway that involves lactate dehydrogenase, therecombinant microorganism produces decreased amounts of lactate incomparison to microorganisms that do not have attenuated endogenouslactate dehydrogenase gene expression. Decrease in the amount of lactateproduced can be measured by routine assays known to one of skill in theart. The amount of lactate reduction is at least about 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% ascompared when no molecular manipulations are done.

The activity of lactate dehydrogenase can also be decreased by othermolecular manipulations of the enzyme. The decrease of enzyme activitycan be any amount of reduction of specific activity or total activity ascompared to when no manipulation has been effectuated. In someinstances, the decrease of enzyme activity is decreased by at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99%.

Accordingly, in some cases, attenuation of the activity of theendogenous lactate dehydrogenase gene results in more carbon flux intothe mevalonate dependent biosynthetic pathway in comparison tomicroorganisms that do not have attenuated endogenous lactatedehydrogenase gene expression.

Pathways Involving Malic Enzyme

Malic enzyme (in E. coli, encoded by the sfcA and maeB genes) is ananaplerotic enzyme that catalyzes the conversion of malate into pyruvate(using NAD+ or NADP+) by the following equation:(S)-malate+NAD(P)⁺

pyruvate+CO₂+NAD(P)H

Thus, the two substrates of this enzyme are (S)-malate and NAD(P)⁺,whereas its 3 products are pyruvate, CO₂, and NADPH.

Expression of the NADP-dependent malic enzyme (MaeB) (Iwikura, M. et al.1979. J. Biochem. 85: 1355-1365) can help increase mevalonate, isoprene,isoprenoid precursors and isoprenoids yield by 1) bringing carbon fromthe TCA cycle back to pyruvate, direct precursor of acetyl-CoA, itselfdirect precursor of the mevalonate pathway and 2) producing extra NADPHwhich could be used in the HMG-CoA reductase reaction (Oh, M K et al.(2002) J Biol. Chem. 277: 13175-13183; Bologna, F. et al. (2007) J Bact.189:5937-5946).

As such, more starting substrate (pyruvate or acetyl-CoA) for thedownstream production of mevalonate, isoprene, isoprenoid precursors andisoprenoids can be achieved by modulating, such as increasing, theactivity and/or expression of malic enzyme. The NADP-dependent malicenzyme gene can be an endogenous gene. One non-limiting way toaccomplish this is by replacing the endogenous NADP-dependent malicenzyme gene promoter with a synthetic constitutively expressingpromoter. Another non-limiting way to increase enzyme activity is byusing one or more heterologous nucleic acids encoding an NADP-dependentmalic enzyme polypeptide. One of skill in the art can monitor theexpression of maeB RNA during fermentation or culturing using readilyavailable molecular biology techniques.

Accordingly, in some embodiments, the recombinant microorganism producesincreased amounts of pyruvate in comparison to microorganisms that donot have increased expression of an NADP-dependent malic enzyme gene. Insome aspects, increasing the activity of an NADP-dependent malic enzymegene results in more carbon flux into the mevalonate dependentbiosynthetic pathway in comparison to microorganisms that do not haveincreased NADP-dependent malic enzyme gene expression.

Increase in the amount of pyruvate produced can be measured by routineassays known to one of skill in the art. The amount of pyruvate increasecan be at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% as compared when no molecular manipulationsare done.

The activity of malic enzyme can also be increased by other molecularmanipulations of the enzyme. The increase of enzyme activity can be anyamount of increase of specific activity or total activity as compared towhen no manipulation has been effectuated. In some instances, theincrease of enzyme activity is at least about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

Pathways Involving Pyruvate Dehydrogenase Complex

The pyruvate dehydrogenase complex, which catalyzes the decarboxylationof pyruvate into acetyl-CoA, is composed of the proteins encoded by thegenes aceE, aceF and lpdA. Transcription of those genes is regulated byseveral regulators. Thus, one of skill in the art can increaseacetyl-CoA by modulating the activity of the pyruvate dehydrogenasecomplex. Modulation can be to increase the activity and/or expression(e.g., constant expression) of the pyruvate dehydrogenase complex. Thiscan be accomplished by different ways, for example, by placing a strongconstitutive promoter, like PL.6(aattcatataaaaaacatacagataaccatctgcggtgataaattatctctggcggtgttgacataaataccactggcggtgatactgagcacatcagcaggacgcactgaccaccatgaaggtg (SEQ ID NO:113), lambda promoter,GenBank NC_001416), in front of the operon or using one or moresynthetic constitutively expressing promoters.

Accordingly, in one aspect, the activity of pyruvate dehydrogenase ismodulated by increasing the activity of one or more genes of thepyruvate dehydrogenase complex consisting of (a) pyruvate dehydrogenase(E1), (b) dihydrolipoyl transacetylase, and (c) dihydrolipoyldehydrogenase. It is understood that any one, two or three of thesegenes can be manipulated for increasing activity of pyruvatedehydrogenase. In another aspect, the activity of the pyruvatedehydrogenase complex can be modulated by attenuating the activity of anendogenous pyruvate dehydrogenase complex repressor gene, furtherdetailed below. The activity of an endogenous pyruvate dehydrogenasecomplex repressor can be attenuated by deletion of the endogenouspyruvate dehydrogenase complex repressor gene.

In some cases, one or more genes of the pyruvate dehydrogenase complexare endogenous genes. Another way to increase the activity of thepyruvate dehydrogenase complex is by introducing into the microorganismone or more heterologous nucleic acids encoding one or more polypeptidesfrom the group consisting of (a) pyruvate dehydrogenase (E1), (b)dihydrolipoyl transacetylase, and (c) dihydrolipoyl dehydrogenase.

By using any of these methods, the recombinant microorganism can produceincreased amounts of acetyl-CoA in comparison to microorganisms whereinthe activity of pyruvate dehydrogenase is not modulated. Modulating theactivity of pyruvate dehydrogenase can result in more carbon flux intothe mevalonate dependent biosynthetic pathway in comparison tomicroorganisms that do not have modulated pyruvate dehydrogenaseexpression.

Exemplary Combinations of Mutations

It is understood that for any of the enzymes and/or enzyme pathwaysdescribed herein, molecular manipulations that modulate any combination(two, three, four, five, six, or more) of the enzymes and/or enzymepathways described herein is expressly contemplated.

In one embodiment, for exemplary representation only, and for ease ofthe recitation of the combinations, citrate synthase (gltA) isdesignated as A, phosphotransacetylase (ptaB) is designated as B,acetate kinase (ackA) is designated as C, lactate dehydrogenase (ldhA)is designated as D, malic enzyme (sfcA or maeB) is designated as E,pyruvate decarboxylase (aceE, aceF, and/or lpdA) is designated as F,6-phosphogluconolactonase (ybhE) is designated as G, phosphoenolpyruvatecarboxylase (ppl) is designated as H, acetyltransferase (such as YfiQ)is designated as I, and deacetylase (such as CobB) is designated as J.As discussed above, aceE, aceF, and/or lpdA enzymes of the pyruvatedecarboxylase complex can be used singly, or two of three enzymes, orthree of three enzymes for increasing pyruvate decarboxylase activity.Accordingly, in this exemplary embodiment, for combinations of any twoof the enzymes A-J, non-limiting combinations that can be used are: AB,AC, AD, AE, AF, AG, AH, AI, AJ, BC, BD, BE, BF, BG, BH, BI, BJ, CD, CE,CF, CG, CH, CI, CJ, DE, DF, DG, DH, DI, DJ, EF, EG, EH, EI, EJ, GH, GI,GJ, HI, HJ, and IJ. For combinations of any three of the enzymes A-J,non-limiting combinations that can be used are: ABC, ABD, ABE, ABF, ABG,ABH, ABI, ABJ, BCD, BCE, BCF, BCG, BCH, BCI, BCJ, CDE, CDF, CDG, CDH,CDI, CDJ, DEF, DEG, DEH, DEI, DEJ, ACD, ACE, ACF, ACG, ACH, ACI, ACJ,ADE, ADF, ADG, ADH, ADI, ADJ, AEF, AEG, AEH, AEI, AEJ, BDE, BDF, BDG,BDH, BDI, BDJ, BEF, BEG, BEH, BEI, BEJ, CEF, CEG, CEH, CEI, CEJ, CFG,CFH, CFI, CFJ, CGH, CGI, and CGJ. For combinations of any four of theenzymes A-J, non-limiting combinations that can be used are: ABCD, ABCE,ABCF, ABCG, ABCH, ABCI, ABCJ, ABDE, ABDF, ABDG, ABDH, ABDI, ABDJ, ABEF,ABEG, ABEH, ABEI, ABEJ, BCDE, BCDF, BCDG, BCDH, BCDI, BCDJ, CDEF, CDEG,CDEH, CDEI, CDEJ, ACDE, ACDF, ACDG, ACDH, ACDI, ACDJ, ACEF, ACEG, ACEH,ACEI, ACEJ, BCEF, BDEF, BGEF, BHEF, BIEF, BJEF, and ADEF. Forcombinations of any five of the enzymes A-J, non-limiting combinationsthat can be used are: ABCDE, ABCDF, ABCDG, ABCDH, ABCDI, ABCDJ, ABDEF,ABDEG, ABDEH, ABDEI, ABDEJ, BCDEF, BCDEG, BCDEH, BCDEI, BCDEJ, ACDEF,ACDEG, ACEDH, ACEDI, ACEDJ, ABCEF, ABCEG, ABCEH, ABCEI, and ABCEJ. Forcombinations of any six of the enzymes A-J, non-limiting combinationsthat can be used are: ABCDEF, ABCDEG, ABCDEH, ABCDEI, ABCDEJ, BCDEFG,BCDEFH, BCDEFI, BCDEFJ, CDEFGH, CDEFGI, and CDEFGJ. For combinations ofany seven of the enzymes A-J, non-limiting combinations that can be usedare: ABCDEFG, ABCDEFH, ABCDEFI, ABCDEFJ, BCDEFGH, BCDEFGI, and BCDEFGJ.For combinations of any eight of the enzymes A-J, non-limitingcombinations that can be used are: ABCDEFGH, ABCDEFGI, and ABCDEFGJ. Forcombinations of any nine of the enzymes A-J, non-limiting combinationsthat can be used are: ABCDEFGHI and ABCDEFGHJ. In another aspect, allten enzyme combinations are used ABCDEFGHIJ.

In other embodiments, any of the mutations described herein can becombined and expressed in recombinant cells for use in effectuating theimproved/enhanced/increased production of mevalonate, isoprene,isoprenoids, isoprenoid precursors, and/or acetyl-CoA-derived products.

Accordingly, the recombinant microorganism as described herein canachieve increased mevalonate production that is increased compared tomicroorganisms that are not grown under conditions of tri-carboxylicacid (TCA) cycle activity, wherein metabolic carbon flux in therecombinant microorganism is directed towards mevalonate production bymodulating the activity of one or more enzymes from the group consistingof (a) citrate synthase, (b) phosphotransacetylase and/or acetatekinase, (c) lactate dehydrogenase, (d) malic enzyme, (e) pyruvatedecarboxylase complex (f) acetyltransferases (such as YfiQ), and (g)deacetylases (such as CobB).

Other Regulators and Factors for Increased Production

Other molecular manipulations can be used to increase the flow of carbontowards mevalonate production. One method is to reduce, decrease oreliminate the effects of negative regulators for pathways that feed intothe mevalonate pathway. For example, in some cases, the genes aceEF-lpdAare in an operon, with a fourth gene upstream pdhR. PdhR is a negativeregulator of the transcription of its operon. In the absence ofpyruvate, it binds its target promoter and represses transcription. Italso regulates ndh and cyoABCD in the same way (Ogasawara, H. et al.2007. J. Bact. 189:5534-5541). In one aspect, deletion of pdhR regulatorcan improve the supply of pyruvate, and hence the production ofmevalonate, isoprene, isoprenoid precursors, and isoprenoids.

In other aspects, the introduction of 6-phosphogluconolactonase (PGL)into microorganisms (such as various E. coli strains) which havedecreased PGL or lack PGL can be used to improve production ofacetyl-CoA-derived products, mevalonate, isoprene, isoprenoidprecursors, and isoprenoids. PGL may be introduced using chromosomalintegration or extra-chromosomal vehicles, such as plasmids. In yetother aspects, PGL may be deleted from the genome of cells (for example,microorganisms, such as various E. coli strains) which express a PGL toimprove production of mevalonate and/or isoprene. In another aspect, aheterologous nucleic acid encoding a PGL polypeptide can be expressed ina cell which does not endogenously express PGL. In some aspects,deletion of PGL results in any of about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100%, inclusive, such as any values in between thesepercentages, higher percent yield of isoprene in comparison tomicroorganisms that express PGL. In other aspects, deletion of PGLresults in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%, inclusive, including any values in between these percentages,higher instantaneous percent yield of isoprene in comparison tomicroorganisms that express PGL. In other aspects, deletion of PGLresults in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%, inclusive, including any values in between these percentages,higher cell productivity index for isoprene in comparison tomicroorganisms that express PGL. In other aspects, deletion of PGLresults in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%, inclusive, including any values in between these percentages,higher volumetric productivity of isoprene in comparison tomicroorganisms that express PGL. In other aspects, deletion of PGLresults in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%, inclusive, including any values in between these percentages,higher peak specific productivity of isoprene in comparison tomicroorganisms that express PGL. In some aspects the deletion of PGLresults in peak specific productivity being maintained for a longerperiod of time in comparison to microorganisms that express PGL.

In another aspect, modulation of phosphoenolpyruvate carboxylase (ppc inE. coli) gene expression can be used to improve production ofmevalonate, isoprene, isoprenoid precursor molecules, isoprenoids,and/or acetyl-CoA derived products in any of the cells disclosed herein.In one aspect, the gene expression of phosphoenolpyruvate carboxylasecan be decreased by replacing the promoter sequence of the ppc gene withanother promoter that results in decreased ppc gene expression incomparison to wild type cells. In some aspects, ppc gene expression canbe decreased by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100%, inclusive, including any values in between thesepercentages, in comparison to wild type cells. In some aspects,decreased expression of phosphoenolpyruvate carboxylase results in anyof about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%,inclusive, such as any values in between these percentages, higherpercent yield of isoprene in comparison to microorganisms that expressphosphoenolpyruvate carboxylase at wild type levels. In other aspects,decreased expression of phosphoenolpyruvate carboxylase results in anyof about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%,inclusive, including any values in between these percentages, higherinstantaneous percent yield of isoprene in comparison to microorganismsthat express phosphoenolpyruvate carboxylase at wild type levels. Inother aspects, decreased expression of phosphoenolpyruvate carboxylaseresults in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%, inclusive, including any values in between these percentages,higher cell productivity index for isoprene in comparison tomicroorganisms that express phosphoenolpyruvate carboxylase at wild typelevels. In other aspects, decreased expression of phosphoenolpyruvatecarboxylase results in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100%, inclusive, including any values in between thesepercentages, higher volumetric productivity of isoprene in comparison tomicroorganisms that express phosphoenolpyruvate carboxylase at wild typelevels. In other aspects, decreased expression of phosphoenolpyruvatecarboxylase results in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100%, inclusive, including any values in between thesepercentages, higher peak specific productivity of isoprene in comparisonto microorganisms that express phosphoenolpyruvate carboxylase at wildtype levels. In some aspects decreased expression of phosphoenolpyruvatecarboxylase results in peak specific productivity being maintained for alonger period of time in comparison to microorganisms that expressphosphoenolpyruvate carboxylase at wild type levels.

In another aspect, modulation of the inhibitor of RssB activity duringmagnesium starvation (iraMin E. coli) gene expression can be used toimprove production of mevalonate, isoprene, isoprenoid precursormolecules, isoprenoids, and/or acetyl-CoA derived products can used inany of the cells disclosed herein. In one aspect, the gene expression ofiraM can be increased by replacing the promoter sequence of the iraMgene with another promoter that results in increased iraM geneexpression in comparison to wild type cells. In some aspects, iraM geneexpression can be increased by any of about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100%, inclusive, including any values in betweenthese percentages, in comparison to wild type cells. In some aspects,increased expression of the iraM gene results in any of about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive, such as anyvalues in between these percentages, higher percent yield of isoprene incomparison to microorganisms that express the iraM gene at wild typelevels. In other aspects, increased expression of the iraM gene resultsin any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%,inclusive, including any values in between these percentages, higherinstantaneous percent yield of isoprene in comparison to microorganismsthat express the iraM gene at wild type levels. In other aspects,increased expression of the iraM gene results in any of about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive, including anyvalues in between these percentages, higher cell productivity index forisoprene in comparison to microorganisms that express the iraM gene atwild type levels. In other aspects, increased expression of the iraMgene results in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100%, inclusive, including any values in between thesepercentages, higher volumetric productivity of isoprene in comparison tomicroorganisms that express the iraM gene at wild type levels. In otheraspects, increased expression of the iraM gene results in any of about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive,including any values in between these percentages, higher peak specificproductivity of isoprene in comparison to microorganisms that expressthe iraM gene at wild type levels. In some aspects increased expressionof the iraM gene results in peak specific productivity being maintainedfor a longer period of time in comparison to microorganisms that expressthe iraM gene at wild type levels.

In another aspect, modulation of the AcrA component of the multidrugefflux pump acrAB-TolC (the acrA gene in E. coli) gene expression can beused to improve production of mevalonate, isoprene, isoprenoid precursormolecules, isoprenoids, and/or acetyl-CoA derived products in any of thecells disclosed herein. In one aspect, the gene expression of acrA canbe decreased by replacing the promoter sequence of the acrA gene withanother promoter that results in decreased acrA gene expression incomparison to wild type cells. In some aspects, acrA gene expression canbe decreased by any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100%, inclusive, including any values in between thesepercentages, in comparison to wild type cells. In another aspect,expression of acrA can be completely abolished, such as by deleting, theacrA gene in the genome of the cell, so that it no longer produces afunctional acrA protein. In some aspects, deletion or decreasedexpression of the acrA gene results in any of about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100%, inclusive, such as any values inbetween these percentages, higher percent yield of isoprene incomparison to microorganisms that express the acrA gene at wild typelevels. In other aspects, deletion or decreased expression of the acrAgene results in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100%, inclusive, including any values in between thesepercentages, higher instantaneous percent yield of isoprene incomparison to microorganisms that express the acrA gene at wild typelevels. In other aspects, deletion or decreased expression of the acrAgene results in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100%, inclusive, including any values in between thesepercentages, higher cell productivity index for isoprene in comparisonto microorganisms that express the acrA gene at wild type levels. Inother aspects, deletion or decreased expression of the acrA gene resultsin any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%,inclusive, including any values in between these percentages, highervolumetric productivity of isoprene in comparison to microorganisms thatexpress the acrA gene at wild type levels. In other aspects, deletion ordecreased expression of the acrA gene results in any of about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive, including anyvalues in between these percentages, higher peak specific productivityof isoprene in comparison to microorganisms that express the acrA geneat wild type levels. In some aspects deletion or decreased expression ofthe acrA gene results in peak specific productivity being maintained fora longer period of time in comparison to microorganisms that express theacrA gene at wild type levels.

In another aspect, modulation of FNR DNA binding transcriptionalregulator (FNR) gene expression can be used to improve production ofmevalonate, isoprene, isoprenoid precursor molecules, isoprenoids,and/or acetyl-CoA derived products in any of the cells disclosed herein.In one aspect, the gene expression of FNR can be increased by replacingthe promoter sequence of the gene which encodes FNR with anotherpromoter that results in increased FNR expression in comparison to wildtype cells. In other aspects, a heterologous nucleic acid encoding FNRcan be expressed in a cell that does not endogenously express FNR. Insome aspects, FNR expression can be increased by any of about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive, including anyvalues in between these percentages, in comparison to wild type cells orcells that do not endogenously express FNR. In some aspects, increasedFNR expression results in any of about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100%, inclusive, such as any values in between thesepercentages, higher percent yield of isoprene in comparison to wild typecells or cells that do not endogenously express FNR. In other aspects,increased FNR expression results in any of about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100%, inclusive, including any values inbetween these percentages, higher instantaneous percent yield ofisoprene in comparison to in comparison to wild type cells or cells thatdo not endogenously express FNR. In other aspects, increased FNRexpression results in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100%, inclusive, including any values in between thesepercentages, higher cell productivity index for isoprene in comparisonto wild type cells or cells that do not endogenously express FNR. Inother aspects, increased FNR expression results in any of about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, inclusive, includingany values in between these percentages, higher volumetric productivityof isoprene in comparison to wild type cells or cells that do notendogenously express FNR. In other aspects, increased FNR expressionresults in any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%, inclusive, including any values in between these percentages,higher peak specific productivity of isoprene in comparison to wild typecells or cells that do not endogenously express FNR. In some aspectsincreased FNR expression results in peak specific productivity beingmaintained for a longer period of time in comparison to wild type cellsor cells that do not endogenously express FNR.

Exemplary Host Cells

Any microorganism or progeny thereof that can be used to heterologouslyexpress one or more genes (e.g., recombinant host cell) and can beengineered such that the expression of the nucleic acids and/or activityof the acetylating protein(s) produced by said cell is modulated, can beused as described herein for increased production of mevalonate,isoprene, isoprenoid precursor molecules, isoprenoids, and/or acetyl-CoAderived products.

Bacteria cells, including gram positive or gram negative bacteria can beused to express any of the nucleic acids described above. In particular,nucleic acids can be expressed in any one of P. citrea, B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S.albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., and P.alcaligenes cells. In some aspects, the host cell can be a Lactobacilisspp., such as Lactobacillus lactis or a Lactobacillus plantarum.

There are numerous types of anaerobic cells that can be used as hostcells in the compositions and methods of the present invention. In oneaspect of the invention, the cells described in any of the compositionsor methods described herein are obligate anaerobic cells and progenythereof. Obligate anaerobes typically do not grow well, if at all, inconditions where oxygen is present. It is to be understood that a smallamount of oxygen may be present, that is, there is some tolerance levelthat obligate anaerobes have for a low level of oxygen. In one aspect,obligate anaerobes engineered to produce mevalonate, isoprene,isoprenoid precursor molecules, isoprenoids, and/or acetyl-CoA derivedproducts can serve as host cells for any of the methods and/orcompositions described herein and are grown under substantiallyoxygen-free conditions, wherein the amount of oxygen present is notharmful to the growth, maintenance, and/or fermentation of theanaerobes.

In another aspect of the invention, the host cells described and/or usedin any of the compositions or methods described herein are facultativeanaerobic cells and progeny thereof. Facultative anaerobes can generatecellular ATP by aerobic respiration (e.g., utilization of the TCA cycle)if oxygen is present. However, facultative anaerobes can also grow inthe absence of oxygen. This is in contrast to obligate anaerobes whichdie or grow poorly in the presence of greater amounts of oxygen. In oneaspect, therefore, facultative anaerobes can serve as host cells for anyof the compositions and/or methods provided herein and can be engineeredto produce mevalonate, isoprene, isoprenoid precursor molecules,isoprenoids, and/or acetyl-CoA derived products. Facultative anaerobichost cells can be grown under substantially oxygen-free conditions,wherein the amount of oxygen present is not harmful to the growth,maintenance, and/or fermentation of the anaerobes, or can bealternatively grown in the presence of greater amounts of oxygen.

The host cell can additionally be a filamentous fungal cell and progenythereof. (See, e.g., Berka & Barnett, Biotechnology Advances, (1989),7(2):127-154). In some aspects, the filamentous fungal cell can be anyof Trichoderma longibrachiatum, T. viride, T. koningii, T harzianum,Penicillium sp., Humicola insolens, H. lanuginose, H. grisea,Chrysosporium sp., C. lucknowense, Gliocladium sp., Aspergillus sp.,such as A. oryzae, A. niger, A sojae, A. japonicus, A. nidulans, or A.awamori, Fusarium sp., such as F. roseum, F. graminum F. cerealis, F.oxysporuim, or F. venenatum, Neurospora sp., such as N. crassa, Hypocreasp., Mucor sp., such as M. miehei, Rhizopus sp. or Emericella sp. Insome aspects, the fungus is A. nidulans, A. awamori, A. oryzae, A.aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F. oxysporum,or F. solani. In certain embodiments, plasmids or plasmid components foruse herein include those described in U.S. patent pub. No. US2011/0045563.

The host cell can also be a yeast, such as Saccharomyces sp.,Schizosaccharomyces sp., Pichia sp., or Candida sp. In some aspects, theSaccharomyces sp. is Saccharomyces cerevisiae (See, e.g., Romanos etal., Yeast, (1992), 8(6):423-488). In certain embodiments, plasmids orplasmid components for use herein include those described in U.S. Pat.No. 7,659,097 and U.S. patent pub. No. US 2011/0045563.

The host cell can additionally be a species of algae, such as a greenalgae, red algae, glaucophytes, chlorarachniophytes, euglenids,chromista, or dinoflagellates. (See, e.g., Saunders & Warmbrodt, “GeneExpression in Algae and Fungi, Including Yeast,” (1993), NationalAgricultural Library, Beltsville, Md.). In certain embodiments, plasmidsor plasmid components for use herein include those described in U.S.Patent Pub. No. US 2011/0045563. In some aspects, the host cell is acyanobacterium, such as cyanobacterium classified into any of thefollowing groups based on morphology: Chlorococcales, Pleurocapsales,Oscillatoriales, Nostocales, or Stigonematales (See, e.g., Lindberg etal., Metab. Eng., (2010) 12(1):70-79). In certain embodiments, plasmidsor plasmid components for use herein include those described in U.S.patent pub. No. US 2010/0297749; US 2009/0282545 and Intl. Pat. Appl.No. WO 2011/034863.

E. coli host cells that comprise one or more nucleic acids encoding oneor more acetylating proteins, wherein said cells have been engineeredsuch that the expression of the nucleic acids and/or activity of theacetylating protein(s) is modulated can be used to express one or moreupper MVA pathway polypeptides, such as any of the upper MVA pathwaypolypeptides described herein. In some aspects, E. coli host cells canbe used to express one or more mvaE and MvaS polypeptides in thecompositions and methods described herein. In one aspect, the host cellis a recombinant cell of an Escherichia coli (E. coli) strain, orprogeny thereof, capable of producing mevalonate that expresses one ormore nucleic acids encoding upper MVA pathway polypeptides. The E. colihost cells (such as those cells that have been engineered as describedherein) can produce mevalonate in amounts, peak titers, and cellproductivities greater than that of the same cells lacking one or moreheterologously expressed nucleic acids encoding upper MVA pathwaypolypeptides and which do not comprise one or more nucleic acidsencoding one or more acetylating proteins, wherein said cells have beenengineered such that the expression of the nucleic acids and/or activityof the acetylating protein(s) is modulated. In addition, the one or moreheterologously expressed nucleic acids encoding upper MVA pathwaypolypeptides in E. coli can be chromosomal copies (e.g., integrated intothe E. coli chromosome). In another aspect, the one or moreheterologously expressed nucleic acids encoding mvaE and MvaSpolypeptides in E. coli can be chromosomal copies (e.g., integrated intothe E. coli chromosome). In other aspects, the E. coli cells are inculture.

Exemplary Vectors

One of skill in the art will recognize that expression vectors aredesigned to contain certain components which optimize gene expressionfor certain host strains. Such optimization components include, but arenot limited to origin of replication, promoters, and enhancers. Thevectors and components referenced herein are described for exemplarypurposes and are not meant to narrow the scope of the invention.

Suitable vectors can be used for any of the compositions and methodsdescribed herein. For example, suitable vectors can be used to optimizethe expression of one or more copies of a gene encoding an MVA pathwaypolypeptide, an isoprene synthase, and/or a polyprenyl pyrophosphatesynthase in anaerobes. In some aspects, the vector contains a selectivemarker. Examples of selectable markers include, but are not limited to,antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin,carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin,or chloramphenicol) and/or nucleic acids that confer a metabolicadvantage, such as a nutritional advantage on the host cell. In someaspects, one or more copies of an upper MVA pathway polypeptide, anisoprene synthase, a polyprenyl pyrophosphate synthase, and/or one ormore MVA pathway polypeptide nucleic acid(s) integrate into the genomeof host cells without a selective marker.

Any one of the vectors characterized herein or used in the Examples ofthe present disclosure can be used.

Exemplary Transformation Methods

Nucleic acids encoding one or more copies of an upper MVA pathwaypolypeptide, isoprene synthase, lower MVA pathway polypeptides, and/orphosphoketolase can be inserted into a microorganism using suitabletechniques. Additionally, isoprene synthase, IDI, DXP pathway, and/orpolyprenyl pyrophosphate synthase nucleic acids or vectors containingthem can be inserted into a host cell (e.g., a plant cell, a fungalcell, a yeast cell, or a bacterial cell described herein) using standardtechniques for introduction of a DNA construct or vector into a hostcell, such as transformation, electroporation, nuclear microinjection,transduction, transfection (e.g., lipofection mediated or DEAE-Dextrinmediated transfection or transfection using a recombinant phage virus),incubation with calcium phosphate DNA precipitate, high velocitybombardment with DNA-coated microprojectiles, and protoplast fusion.General transformation techniques are known in the art (See, e.g.,Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds.)Chapter 9, 1987; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor, 1989; and Campbell et al., Curr.Genet. 16:53-56, 1989). The introduced nucleic acids can be integratedinto chromosomal DNA or maintained as extrachromosomal replicatingsequences. Transformants can be selected by any method known in the art.Suitable methods for selecting transformants are described inInternational Publication No. WO 2009/076676, U.S. patent applicationSer. No. 12/335,071 (US Publ. No. 2009/0203102), WO 2010/003007, USPubl. No. 2010/0048964, WO 2009/132220, and US Publ. No. 2010/0003716,the disclosures of which are incorporated by reference herein.

Exemplary Cell Culture Media

As used herein, the terms “minimal medium” or “minimal media” refer togrowth medium containing the minimum nutrients possible for cell growth,generally, but not always, without the presence of one or more aminoacids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids).Minimal medium typically contains: (1) a carbon source for microorganism(e.g., bacterial, algal, or fungal cell) growth; (2) various salts,which can vary among microorganism (e.g., bacterial, algal, or fungal)species and growing conditions; and (3) water. The carbon source canvary significantly, from simple sugars like glucose to more complexhydrolysates of other biomass, such as yeast extract, as discussed inmore detail below. The salts generally provide essential elements suchas magnesium, nitrogen, phosphorus, and sulfur to allow the cells tosynthesize proteins and nucleic acids. Minimal medium can also besupplemented with selective agents, such as antibiotics, to select forthe maintenance of certain plasmids and the like. For example, if amicroorganism is resistant to a certain antibiotic, such as ampicillinor tetracycline, then that antibiotic can be added to the medium inorder to prevent cells lacking the resistance from growing. Medium canbe supplemented with other compounds as necessary to select for desiredphysiological or biochemical characteristics, such as particular aminoacids and the like.

Any minimal medium formulation can be used to cultivate the host cells.Exemplary minimal medium formulations include, for example, M9 minimalmedium and TM3 minimal medium. Each liter of M9 minimal medium contains(1) 200 mL sterile M9 salts (64 g Na₂HPO₄-7H₂O, 15 g KH₂PO₄, 2.5 g NaCl,and 5.0 g NH₄Cl per liter); (2) 2 mL of 1 M. MgSO₄ (sterile); (3) 20 mLof 20% (w/v) glucose (or other carbon source); and (4) 100 μl of 1 M.CaCl₂) (sterile). Each liter of TM3 minimal medium contains (1) 13.6 gK₂HPO₄; (2) 13.6 g KH₂PO₄; (3) 2 g MgSO₄*7H₂O; (4) 2 g Citric AcidMonohydrate; (5) 0.3 g Ferric Ammonium Citrate; (6) 3.2 g (NH₄)₂SO₄; (7)0.2 g yeast extract; and (8) 1 mL of 1000× Trace Elements solution; pHis adjusted to ˜6.8 and the solution is filter sterilized. Each liter of1000× Trace Elements contains: (1) 40 g Citric Acid Monohydrate; (2) 30g MnSO₄*H₂O; (3) 10 g NaCl; (4) 1 g FeSO₄*7H₂O; (4) 1 g CoCl₂*6H₂O; (5)1 g ZnSO₄*7H₂O; (6) 100 mg CuSO₄*5H₂O; (7) 100 mg H₃BO₃; and (8) 100 mgNaMoO₄*2H₂O; pH is adjusted to ˜3.0.

An additional exemplary minimal media includes (1) potassium phosphateK₂HPO₄, (2) Magnesium Sulfate MgSO₄*7H₂O, (3) citric acid monohydrateC₆H₈O₇*H₂O, (4) ferric ammonium citrate NH₄FeC₆H₅O₇, (5) yeast extract(from biospringer), (6) 1000× Modified Trace Metal Solution, (7)sulfuric acid 50% w/v, (8) foamblast 882 (Emerald PerformanceMaterials), and (9) Macro Salts Solution 3.36 mL All of the componentsare added together and dissolved in deionized H₂O and then heatsterilized. Following cooling to room temperature, the pH is adjusted to7.0 with ammonium hydroxide (28%) and q.s. to volume. Vitamin Solutionand spectinomycin are added after sterilization and pH adjustment.

Any carbon source can be used to cultivate the host cells. The term“carbon source” refers to one or more carbon-containing compoundscapable of being metabolized by a host cell or organism. For example,the cell medium used to cultivate the host cells can include any carbonsource suitable for maintaining the viability or growing the host cells.In some aspects, the carbon source is a carbohydrate (such asmonosaccharide, disaccharide, oligosaccharide, or polysaccharides), orinvert sugar (e.g., enzymatically treated sucrose syrup).

In some aspects, the carbon source includes yeast extract or one or morecomponents of yeast extract. In some aspects, the concentration of yeastextract is 0.1% (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06%(w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01%(w/v) yeast extract. In some aspects, the carbon source includes bothyeast extract (or one or more components thereof) and another carbonsource, such as glucose.

Exemplary monosaccharides include glucose and fructose; exemplaryoligosaccharides include lactose and sucrose, and exemplarypolysaccharides include starch and cellulose. Exemplary carbohydratesinclude C6 sugars (e.g., fructose, mannose, galactose, or glucose) andC5 sugars (e.g., xylose or arabinose).

Exemplary Cell Culture Conditions

Materials and methods suitable for the maintenance and growth of therecombinant cells of the invention are described infra, e.g., in theExamples section. Other materials and methods suitable for themaintenance and growth of cell (e.g. bacterial, fungal, algal) culturesare well known in the art. Exemplary techniques can be found inInternational Publication No. WO 2009/076676, U.S. Publ. No.2009/0203102, WO 2010/003007, US Publ. No. 2010/0048964, WO 2009/132220,US Publ. No. 2010/0003716, Manual of Methods for General BacteriologyGerhardt et al., eds), American Society for Microbiology, Washington,D.C. (1994) or Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989) Sinauer Associates, Inc.,Sunderland, Mass.

Standard cell culture conditions can be used to culture the cells (see,for example, WO 2004/033646 and references cited therein). In someaspects, cells are grown and maintained at an appropriate temperature,gas mixture, and pH (such as at about 20° C. to about 37° C., at about6% to about 84% CO₂, and at a pH between about 5 to about 9). In someaspects, cells are grown at 35° C. in an appropriate cell medium. Insome aspects, the pH ranges for fermentation are between about pH 5.0 toabout pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5 to about7.0). Cells can be grown under aerobic, anoxic, or anaerobic conditionsbased on the requirements of the host cells. In addition, more specificcell culture conditions can be used to culture the cells. For example,in some embodiments, the cells (e.g., bacterial cells, such as E. colicells, fungal cells, algal cells) can express one or more heterologousnucleic acids under the control of a strong promoter in a low to mediumcopy plasmid and are cultured at 34° C.

Standard culture conditions and modes of fermentation, such as batch,fed-batch, or continuous fermentation that can be used are described inInternational Publication No. WO 2009/076676, U.S. patent applicationSer. No. 12/335,071 (U.S. Publ. No. 2009/0203102), WO 2010/003007, USPubl. No. 2010/0048964, WO 2009/132220, US Publ. No. 2010/0003716. Batchand Fed-Batch fermentations are common and well known in the art andexamples can be found in Brock, Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition (1989) Sinauer Associates, Inc.

In some aspects, the cells are cultured under limited glucoseconditions. By “limited glucose conditions” is meant that the amount ofglucose that is added is less than or about 105% (such as about 100%,90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%) of the amount of glucosethat is consumed by the cells. In particular aspects, the amount ofglucose that is added to the culture medium is approximately the same asthe amount of glucose that is consumed by the cells during a specificperiod of time. In some aspects, the rate of cell growth is controlledby limiting the amount of added glucose such that the cells grow at therate that can be supported by the amount of glucose in the cell medium.In some aspects, glucose does not accumulate during the time the cellsare cultured. In various aspects, the cells are cultured under limitedglucose conditions for greater than or about 1, 2, 3, 5, 10, 15, 20, 25,30, 35, 40, 50, 60, or 70 hours. In various aspects, the cells arecultured under limited glucose conditions for greater than or about 5,10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, or 100% of the totallength of time the cells are cultured. While not intending to be boundby any particular theory, it is believed that limited glucose conditionscan allow more favorable regulation of the cells.

In some aspects, the cells (such as bacterial, fungal, or algal cells)are grown in batch culture. The cells (such as bacterial, fungal, oralgal cells) can also be grown in fed-batch culture or in continuousculture. Additionally, the cells (such as bacterial, fungal, or algalcells) can be cultured in minimal medium, including, but not limited to,any of the minimal media described above. The minimal medium can befurther supplemented with 1.0% (w/v) glucose, or any other six carbonsugar, or less. Specifically, the minimal medium can be supplementedwith 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5%(w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) glucose.Additionally, the minimal medium can be supplemented 0.1% (w/v) or lessyeast extract. Specifically, the minimal medium can be supplemented with0.1% (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05%(w/v), 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeastextract. Alternatively, the minimal medium can be supplemented with 1%(w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% (w/v), 0.4%(w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) glucose and with 0.1%(w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v),0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast extract.

Exemplary Purification Methods

In some aspects, any of the methods described herein further include astep of recovering the compounds produced. In some aspects, any of themethods described herein further include a step of recovering theisoprene. In some aspects, the isoprene is recovered by absorptionstripping (See, e.g., US Appl. Pub. No. US 2011/0178261 A1, thedisclosure of which is incorporated by reference herein). In someaspects, any of the methods described herein further include a step ofrecovering an isoprenoid. In some aspects, any of the methods describedherein further include a step of recovering the terpenoid or carotenoid.

Suitable purification methods are described in more detail in U.S.Patent Application Publication US2010/0196977 A1, the disclosure ofwhich is incorporated by reference herein.

Exemplary In Vitro Protein Acetylation and Deacetylation Assays

Exemplary acetylation assays are carried out in the presence of bufferand Acetyl-CoA. For example the assay can be carried out in 20 mMbuffer, 100 mM NaCl, 100 μM acetyl-CoA, 20 μM USP in the presence andabsence of 200 μM cAMP. Reactions can initiated by the addition of 20 μMMt-PatA, incubated for 10 min at 22° C., and quenched by boiling in SDSloading dye. Exemplary buffers are provided as follows: sodium acetate(pH 4.0-5.0), MES (pH 6.0), HEPES (pH 7.0), Tris (pH 7.5-8.0),BisTrisPropane (pH 9.0), and Glycine (pH 10.0). Reactions can beanalyzed in parallel with SDS-PAGE and Western blotting with anti-AcLysantibody (Cell Signaling Technology) detected quantitatively bychemiluminescence (LI-COR Biosciences).

Deacetylation assays can be carried out at 22° C. in 20 mM Tris-HCl, pH7.5, 100 mM NaCl, 1 mM β-Nicotinamide adenine dinucleotide (NAD⁺), 20 μMauto-acetylated Mt-PatA H173K mutant in the presence and absence of 200μM cAMP. Reactions can be initiated by addition of 5 μM Rv1151c andterminated at various time points by boiling in SDS loading dye. Samplescan be analyzed in parallel using SDS-PAGE and Western blotting usingSDS-PAGE and Western blotting with anti-Ac-Lys antibodies.

Methods described here are used for monitoring acetylation anddeacetylation of all proteins in bacteria, fungal cells, and algalcells.

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

EXAMPLES Example 1: Construction of Reference and Acetylation ModulatoryStrains

This Example describes the construction of reference strains as well asstrains containing mutations in genes responsible for modulatingintracellular protein acetylation.

I. Construction of Reference Strains

The DNA sequence for the phosphoketolase (PKL) enzyme from Mycoplasmahominis (strain ATCC 23114) (SEQ ID NO:28) was codon optimized forexpression in E. coli (SEQ ID NO:29) and synthesized by LifeTechnologies. The M. hominis phosphoketolase was subcloned into plasmidpEWL1421 (pTrc P. alba ispS (MEA variant)-E. gallinarum phosphoketolase)using the GENEART Seamless Cloning and Assembly Kit (Life Technologies)according to manufacturer's protocol, to yield plasmid pMCS826 (FIG. 13). Primers used were MCS534 (cgctaactgcataaaggaggtaaaaaaac)(SEQ IDNO:32) and MCS535 (gctggagaccgtttaaactttaactagacttta) (SEQ ID NO:33) forthe phosphoketolase, and MCS536 (taaagtctagttaaagtttaaacggtctccagc) (SEQID NO:34) and MCS537 (gtttttttacctcctttatgcagttagcg) (SEQ ID NO:35) forthe plasmid pEWL1421 (FIG. 14 ). The sequence of the DNA between theIspS and phosphoketolase open reading frames on plasmid pMCS826 wasaltered by PCR using primers MCS488 (ttagcgttcaaacggcagaatcgg) (SEQ IDNO:36) and MCS562(ccgtttgaacgctaaAGATACGCGTAACCCCAAGGACGGTAAAatgattagcaaaatctatgatgataaaaagtatctgg) (SEQ ID NO:37). The resulting PCR product was purified andself-ligated using standard techniques to form plasmid pMCS1019 (FIG. 15).

A DNA cassette was created by PCR in a 2-stage process. A˜1.8 kb productwas amplified by PCR using primers MCS504 and MCS516 from theFRT-gb2-CM-FRT template (Genebridges). This PCR product was then used astemplate for a second round of amplification by PCR using primers MCS516and MCS545. This PCR product was integrated into the chromosome ofstrain MD891 (BL21 wt, pgl+t PL.2-mKKDyI::FRT, Gi1.2gltAyhfSFRTPyddVIspAyhfS thiFRTtruncIspA pgl ML,FRT-PL.2-2cis-RBS10000-MVK(burtonii) clone A+t ackA::FRT) using theGenebridges Red/ET Recombination Quick and Easy E. coli gene deletionKit, according to manufacturer's instruction. Hereafter, Gi1.2gltAyhfSFRTPyddVIspAyhfS thiFRTtruncIspA and CTO are used interchangeablyand pgl ML and pgl—are used interchangeably. The resulting strain wasdesignated MCS1015 (MD891+ackA::Cm_pL.2_pta). Sequence analysis ofstrain MCS1015 revealed the insertion of the chloramphenicol antibioticmarker and pL.2 promoter upstream of the phosphotransacetylase (pta)gene. The chloramphenicol antibiotic marker was removed from strainMCS1015 by FLP expression according to manufacturer's instruction tocreate strain MCS1016 (MD891+FRT::ackA::FRT_pL.2_pta). Strain MCS1016was transformed with plasmids pMCS1019 (SEQ ID NO:30) and pMCM1225 (SEQID NO:27) to create isogenic strains MCS1227 (pMCS1019(pTrc_IspS_RBS3_PKL16 [M. hominis]), pMCM1225) and MCS1316 (pMCS1019(pTrc_IspS_RBS3_PKL16 [M. hominis]), pMCM1225). The sequences of theprimers used are shown in Table 1-1.

TABLE 1-1 Primer Sequences MCS504tgtttttttacctcctttgcagtgcgtcctgctgatgtgctcagtatcaccgccag SEQ ID NO: 38tggtatttacgtcaacaccgccagagataatttatcaccgcagatggttatcttaatacgactcactatagggctc MCS516gactcaagatatttcttccatcatgcaaaaaaaaatttgcagtgcatgatgttaat SEQ ID NO: 39caaattaaccctcactaaagggcg MCS545gctggtcagaccgacgctggttccggtagggatcagcataataatacgggacatga SEQ ID NO: 40ttttacctcctttgcagtg

II. Construction of Acetylation Chromosome Mutation Strains

Constructs for deletion of yfiQ and cobB were amplified by colony PCRfrom Keio library clones (Baba et al., Molecular Systems Biology 2Article number: 2006.0008 doi:10.1038/msb4100050). The yfiQ deletionconstruct was amplified from JW2568 (plate 21, G12) using primersMCM1038 and MCM1039. The cobB deletion construct was amplified fromJW1106 (plate 21, A2) using primers MCM1033 and MCM1035. 50 uL reactions(Agilent Herculase II kit; Catalog 600679) were performed according tothe manufacturer's protocol with the following conditions for yfiQ: 95°C., 20 min; (95° C., 20 sec; 55° C., 20 sec; 72° C., 1.5 min)×30; 72°C., 3 min; 4° C. hold and the following conditions for cobB: 95° C., 20min; (95° C., 20 sec; 55° C., 20 sec; 72° C., 1.25 min)×30; 72° C., 3min; 4° C. hold. PCR products were purified according to themanufacturer's protocol (Qiagen QIAquick PCR Purification Kit, Catalog28104) and eluted in 30 uL EB. Primer sequences are shown in Table 1-2.

TABLE 1-2 Primer Sequences MCM1033 aggctgcctcgtcatctctt SEQ ID NO: 41MCM1035 cagaatatcgccactctggg SEQ ID NO: 42 MCM1038 acacgctatctggcaggaaaSEQ ID NO: 43 MCM1039 tttgacaacatcacagtgca SEQ ID NO: 44

Constructs for insertion of constitutive promoters at yfiQ and cobB werecreated in a two-step PCR process. The construct designated KanR_gi1.6was amplified by PCR using primers MCS580 and MCS584 from theFRT-PGK-gb2-neo-FRT template (Genebridges). The PCR product was purifiedusing the QIAquick PCR Purification Kit (Qiagen) and used as a template(10 ng per reaction) in further PCRs. The FRT-kan-FRT-gi1.6-YfiQconstruct was amplified using primers MCM1042 and MCM1043. TheFRT-kan-FRT-gi1.6-CobB construct was amplified using primers MCM1046 andMCM1048. 50 uL reactions (Agilent Herculase II kit; Catalog 600679) wereperformed according to the manufacturer's protocol with the followingconditions: 95° C., 20 min; (95° C., 20 sec; 55° C., 20 sec; 72° C.,1.25 min)×30; 72° C., 3 min; 4° C. hold. PCR products were purifiedaccording to the manufacturer's protocol (Qiagen QIAquick PCRPurification Kit, Catalog 28104) and eluted in 30 uL EB. Primersequences are shown in Table 1-3.

TABLE 1-3 Primer Sequences MCS580 aattaaccctcactaaagggcggc SEQ ID NO: 45MCS584 atattccaccagctatttgttagtgaataaaagtggttgaattatttgct SEQ ID NO: 46caggatgtggcattgtcaagggctaatacgactcactatagggctc gaggaag MCM1042tcacagcagaacagttagaaagcgtttaaaatcattcggtcacttct SEQ ID NO: 47gcgggagaccggtaattaaccctcactaaagggcggc MCM1043cgcgccaattaccgctatcgattaggtcgcagtagtgcttccagtcc SEQ ID NO: 48tcgctgactcatatattccaccagctatttgttagtg MCM1046gcgggaggaatgcgtggtgcggccttcctacatctaaccgattaaa SEQ ID NO: 49caacagaggttgctaattaaccctcactaaagggcggc MCM1048gcgcaggcggcgtttatttttacgaaaacgacttaaccgatgaccc SEQ ID NO: 50cgacgcgacagcatatattccaccagctatttgttagtg

Constructs were introduced into strain DW853, which is strain MD891carrying pRedET-carb. Using standard molecular biology procedures, DW853was generated by electroporation of the pRED/ET plasmid (GeneBridges)into MD891, and subsequent propagation of transformants on solid LBmedium plates containing carbenicillin at a concentration of 50 μg/mL at30° Celsius. Cells containing pRedET-carb were grown in LB+carb50 at 30°C. overnight and then diluted 1:100 into fresh LB+carb50 and cultured at30° C. for 2 hr. 130 uL 10% arabinose was added and cells were culturedat 37° C. for approximately 2 hours. Cells were prepared forelectroporation by washing 3× in one half culture volume iced ddH₂O andresuspended in one tenth culture volume of the same. 100 uL of cellsuspension was combined with 3 uL DNA in a 2 mm electroporation cuvette,electroporated at 25 uFD, 200 ohms, 2.5 kV, and immediately quenchedwith 500 uL LB. Cells were recovered shaking at 37° C. for 3 hrs andthen transformants selected overnight on LB/kan10 plates at 37° C.Transformants were restreaked and then grown in liquid LB/kan10 andfrozen in 30% glycerol. Descriptions of strains are shown in Table 1-4.

TABLE 1-4 Descriptions of Strains Strain Genotype MCM2721 MD891 +YfiQ::FRT-kan-FRT MCM2736 MD891 + CobB::FRT-kan-FRT MCM2740 MD891 +FRT-kan-FRT-gi1.6-YfiQ MCM2742 MD891 + FRT-kan-FRT-gi1.6-CobB (BL21 ATG)

III. Transduction of PL.2-pta

The FRT-cmR-FRT-PL.2-pta locus was moved from strain MCS1015 into strainMCM2721 by transduction, retaining the deletion of ackA. A P1 lysate ofMG1655 was used to create a lysate of MCS1015. 100 uL of this lysate wasmixed with 100 uL of an overnight culture of MCM2721 that hadresuspended in half the culture volume of 10 mM MgCl₂ and 5 mM CaCl₂.The reaction was incubated at 30° C., still for 30 minutes and thenquenched with 100 uL 1M. sodium citrate. 500 uL LB was added and theculture shaken at 37° C. for 1 hr before selecting on LB/cmp5 platesovernight at 37° C. A single colony was restreaked on LB/kan10cmp10. Acolony was grown and frozen as MCM2725 (MD891+FRT-kan-FRT::yfiQFRT-cmp-FRT::PL.2-pta).

IV. Loopouts

The antibiotic markers from the above strains were removed by transientexpression of the FLP recombinase. Plasmid pCP20 (see worldwide webcgsc.biology.yale.edu/Site.php?ID=64621) was electroporated andtransformants selected on LB/carb50 at 30° C. A transformant colony wasgrown in LB/carb50 broth at 30° C. until turbid, then cultured at 37 forseveral hours. Culture was then streaked to LB plates and grown at 37°C. overnight. Single colonies were patched to LB, LB/carb50, LB/kan10and LB/cmp5 (for MCM2725 loopouts). Streaks from LB for coloniessensitive to each antibiotic were grown and frozen in 30% glycerol.Loopout strains are shown in Table 1-5.

TABLE 1-5 Loopout strains Parent strain (markers) Looped out strainMCM2721 (kan) MCM2722 MCM2736 (kan) MCM2754 MCM2740 (kan) MCM2760MCM2742 (kan) MCM2764

V. Isoprene Producing Cells

Plasmids pMCS1019 and pMCM1225 were co-electroporated into above hoststo create isoprene-producing cells. Transformants were selected onLB/carb50spec50 and a single colony was grown in liquid LB/carb50spec50and frozen in 30% glycerol. Isoprene producing cells are shown in Table1-6.

TABLE 1-6 Isoprene Producing Cells Selection Temperature Host IsopreneProducing Cell (degrees centigrade) MCM2722 MCM2728 37 MCM2725 MCM273237 MCM2754 MCM2771 30 MCM2760 MCM2773 30 MCM2764 MCM2775 30

Example 2: Evaluation of yfiQ Deletion in the Small Scale Assay forGrowth Rate and Isoprene Specific Productivity

This example measured isoprene production and growth rate in strainscarrying a deletion of the yfiQ gene.

I. Materials and Methods

LB media, TM3 media without Yeast extract and MgSO₄, 10% Yeast extract,1M MgSO₄, 50% Glucose, 200 mM IPTG, 50 mg/mL Spectinomycin, 50 mg/mLCarbenicillin, Aluminum foil seal, 48-well sterile 5 mL block, breatheeasier sealing membrane, aluminum foil seal, 96-well micro titer plates,96-well glass block purchased from Zinsser Analytic. Agilent 6890 GCequipped with a 5973N Mass spectrometer.

Supplemented TM3 media was prepared by combining TM media, (withoutMgSO₄ and Yeast extract) 1% Glucose, 8 mM MgSO₄, 0.02% Yeast extract andappropriate antibiotics. 2 mL of day culture was started in 48-wellsterile block by inoculating overnight culture in supplemented TM3 mediaat 0.2 optical density (OD). Blocks were sealed with breathe easiermembrane and incubated for 2 hours at 34° C., 600 rpm. After 2 hours ofgrowth, OD was measured at 600 nm in the micro titer plate and cellswere induced with 200 μM IPTG. OD reading was taken every hour after theIPTG induction for 4 hours to determine growth rate. OD was measurementwas done in the micro titer plate at appropriate dilution in the TM3media at 600 nm using a SpectraMax Plus190 (Molecular Devices).

100 μL of isoprene samples were collected in a 96 well glass block at 2,3 and 4 hours after IPTG induction. Glass block was sealed with aluminumfoil and incubated at 34° C. while shaking at 450 rpm, for 30 minutes onthe thermomixer. After 30 minutes, the block was kept in 70° C. waterbath for 2 minutes and isoprene headspace measurement was done in GC/MSto determine specific productivity.

II. Results

FIG. 1A shows growth (OD600) for control wild type yfiQ cells versusyfiQ delete cells over 4 hours while FIG. 1B shows overnight growth(OD600). FIG. 2 shows isoprene specific productivity for control wildtype yfiQ cells versus yfiQ delete cells over 4 hours.

Example 3: Effects of yfiQ Gene Deletion on Isoprene Production inStrains Expressing the Mevalonate Pathway and Isoprene Synthase

This example was performed to evaluate isoprene production using amodified E. coli host (BL21 derived production host MD891) whichexpresses introduced genes from the mevalonate pathway and isoprenesynthase and is grown in fed-batch culture at the 15-L scale. Both hoststrains in this experiment carry a deletion in the gene encoding theacetate kinase (AckA) polypeptide and express an M. hominisphosphoketolase. Additionally, both over express phosphotransacetylase(pta).

These isoprene producing cells were run in the same process. Theperformance metrics of control cells, MCS1227 are compared here to thoseof experimental cells that has been deleted for yfiQ, a lysineacetyltransferase (cell details provided in Table 3-1). The relevantperformance metrics are cumulative isoprene yield on glucose, volumetricproductivity of isoprene and CPI.

TABLE 3-1 Cells used in this example Upper pathway Isoprene synthase/Cell Name Host plasmid Phosphoketolase plasmid MCS1227 CTO pgl- IPTGinducible P_(trc) IPTG inducible (Control) PL.2-2cis- expressing(pTrc_IspS_RBS3_PKL16 RBS 10000- E. gallinarum [M. hominis]) bKKDyl,mvaE, mvaS (pMCS1019, Carb 50) ackA::FRT_pL.2_pta (pMCM1225 Spec 50)MCM2732 MD891 + IPTG inducible P_(trc) IPTG inducible (Experimental -FRT-kan- expressing (pTrc_IspS_RBS3_PKL16 YfiQ deletion) FRT::YfiQ E.gallinarum [M. hominis]) FRT-cmp- mvaE, mvaS (pMCS1019, Carb 50)FRT::PL.2-pta (pMCM1225 Spec 50)

I. Materials and Methods

Medium Recipe (per liter fermentation medium): K₂HPO₄ 7.5 g, MgSO₄*7H₂O2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, 50% sulfuric acid 1.6 mL, 1000× Modified Trace MetalSolution 1 mL. All of the components were added together and dissolvedin Di H₂O. This solution was heat sterilized (123° C. for 20 minutes).The pH was adjusted to 7.0 with ammonium hydroxide (28%) and q.s. tovolume. Glucose 10 g, Vitamin Solution 8 mL, and antibiotics were addedafter sterilization and pH adjustment.

1000× Modified Trace Metal Solution (Per Liter):

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in DiH₂O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution wasq.s. to volume and filter sterilized with a 0.22 micron filter.

Vitamin Solution (Per Liter):

Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic acid 1.0 g,pyridoxine hydrochloride 4.0 g. Each component was dissolved one at atime in Di H₂O, pH was adjusted to 3.0 with HCl/NaOH, and then thesolution was q.s. to volume and filter sterilized with 0.22 micronfilter.

Macro Salt Solution (Per Liter):

MgSO₄*7H₂O 296 g, citric acid monohydrate 296 g, ferric ammonium citrate49.6 g. All components were dissolved in water, q.s. to volume andfilter sterilized with 0.22 micron filter.

Feed Solution (Per Kilogram):

Glucose 0.590 kg, Di H₂O 0.393 kg, K₂HPO₄ 7.4 g, and 100% Foamblast8828.9 g. All components were mixed together and autoclaved. Afterautoclaving the feed solution, nutrient supplements are added to thefeed bottle in a sterile hood. Post sterilization additions to the feedare (per kilogram of feed solution), Macro Salt Solution 5.54 mL,Vitamin Solution 6.55 mL, 1000× Modified Trace Metal Solution 0.82 mL.For a target of 100 μM IPTG: 1.87 mL of a sterile 10 mg/mL solution isadded per kilogram of feed.

This experiment was carried out to monitor isoprene production fromglucose at the desired fermentation pH (7.0) and temperature (34° C.).To start each experiment, the appropriate frozen vial of the E. coliproduction strain was thawed and inoculated into a flask withtryptone-yeast extract (LB) medium and the appropriate antibiotics.After the inoculum grew to an optical density of approximately 1.0,measured at 550 nm (OD₅₅₀), 500 mL was used to inoculate a 15-Lbioreactor and bring the initial tank volume to 5 L.

The inlet gas using to maintain bioreactor backpressure at 0.7 bar gaugeand to provide the oxygen to the production organisms was supplied by inhouse facilities that dilute the inlet gas to a known concentration (7.3to 8.3 vol % oxygen).

The batched media had glucose batched in at 9.7 g/L. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). Asyringe containing a sterile solution of IPTG was added to bring theIPTG concentration to 100 μM when the cells were at an OD₅₅₀ of 6. Oncethe glucose was consumed by the culture, as signaled by a rise in pH,the glucose feed solution was fed to meet metabolic demands at ratesless than or equal to 10 g/min. At a fixed time after dissolved oxygenlimitation was established, the temperature was raised from 34° C. to37° C. over the course of one hour. The fermentation was run long enoughto determine the maximum cumulative isoprene mass yield on glucose,typically a total of 64 hrs elapsed fermentation time (EFT). Table 3-2shows process conditions.

TABLE 3-2 Process Conditions Target [IPTG] after Batched bolus additionTarget [IPTG] in [IPTG] at ~EFT 8 hrs Feed Bottle Strain Used (μM) (μM)(μM) MCS922 1.4 100 100 MCM2732 1.4 100 100

Isoprene, Oxygen, Nitrogen, and Carbon Dioxide levels in the off-gaswere determined independently by a Hiden HPR20 (Hiden Analytical) massspectrometer.

Dissolved Oxygen in the fermentation broth is measured by sanitary,sterilizable probe with an optical sensor provided Hamilton Company.

The citrate, glucose, acetate, and mevalonate concentrations in thefermenter broth was determined in broth samples taken at 4 hourintervals by an HPLC analysis. Concentration in broth samples weredetermined by comparison of the refractive index response versus apreviously generated calibration curve using standard of a knownconcentration.

HPLC information was as follows: System: Waters Alliance 2695; Column:BioRad-Aminex HPX-87H Ion Exclusion Column 300 mm×7.8 mm Catalog#125-0140; Column Temperature: 50° C.; Guard column: BioRad—MicroguardCation H refill 30 mm×4.6 mm Catalog #125-0129; Running buffer: 0.01NH₂SO₄; Running buffer flow rate: 0.6 mL/min; Approximate runningpressure: ˜1100-1200 psi; Injection volume: 20 microliters; Detector:Refractive Index (Knauer K-2301;) Runtime: 26 minutes.

II. Results

Isoprene Productivity Metrics (and EFT when the value was taken) areshown in Table 3-3.

TABLE 3-3 Isoprene Productivity CPI Overall (Total g Isoprene Isopreneisoprene/ Titer Volumetric total Peak (gram Cumulative ProductivitygDCW) at Peak Instantaneous isoprene/ % Yield of at time of Max time ofmax Specific yield of average isoprene on max overall Optical overallProductivity isoprene volume of glucose isoprene Density isoprene (mgisoprene/ on glucose tank broth Strain Name (g/g %) yield (g/L/hr)(A550) yield L/hr/OD) (g/g %) in Liters) MCS1227 18.61 2.00 113.9  3.0337.2 22.6 120.1 (60 hrs) (60 hrs) (32 hrs) (60 hrs) (28 hrs) (40.4hrs)   (60 hrs) MCM2732 19.64 2.34 99.6 3.53 47.0 23.8 140.3 (60 hrs)(60 hrs) (24 hrs) (60 hrs) (28 hrs) (48 hrs) (60 hrs)

Broth concentration of acetate measured in each 15-L fermentation overtime is shown in FIG. 3 . The experimental cells that are deleted foryfiQ (MCM2732) finishes with a lower broth concentration of acetate thanthe control cells that are wild type for yfiQ. The broth concentrationof acetate was determined by HPLC.

Specific Glucose Uptake Rate measured in each 15-L fermentation overtime is shown in FIG. 4 . The experimental cells carrying the yfiQdeletion (MCM2732) consistently shows a higher specific glucose uptakerate than the control cells that are wild type for yfiQ. The lowerconcentration of acetate in the broth is presumably the driver for thehigher specific glucose uptake rate, which in turn drives a highervolumetric productivity (shown in FIG. 5 ). Smoothed specific glucoseuptake rate was calculated using the following formula:Specific Glucose Uptake Rate (g/L/hr/OD)=slope of grams glucose consumedper hour(averaged over 8 hour interval)/broth volume*OD

Volumetric productivity achieved in each 15-L fermentation over time isshown in FIG. 5 . The experimental cells carrying the yfiQ deletion(MCM2732) finishes with a higher volumetric productivity of isoprene onglucose than the control cells that are wild type for yfiQ. The 64 hrpoints were used to populate Table 3-3 above. Volumetric Productivitywas calculated using the following formula:Volumetric productivity(g/L/hr)=[Σ(IspER(t)/1000*68.117)]/[t−t ₀], wherethe summation is from t ₀ to t and where IspER is the isoprene evolutionrate. Tank turnaround time is not factored in.

Cumulative yield of isoprene on glucose achieved in each 15-Lfermentation overtime is shown in FIG. 6 . The experimental cellscarrying the yfiQ deletion (MCM2732) finishes with a higher cumulativeyield of isoprene on glucose than the control cells that are wild typefor yfiQ. The lower broth acetate in for MCM2732 represents less lostcarbon but efficiency gain is more than can be explained by therecapture of the lost acetate carbon. The 64 hr points were used topopulate Table 3-3 above. Overall yield was calculated using thefollowing formula:% wt Yield on glucose=Isoprene total(t)/[(Feed Wt(0)−FeedWt(t)+83.5)*0.59)],

where 0.59 is the wt % of glucose in the glucose feed solution and 83.5is the grams of this feed batched into the fermenter at t=0. Each feedhad its weight % measured independently.

Cell Performance Index (CPI) achieved in each 15-L fermentation overtime is shown in FIG. 7 . The experimental cells carrying the yfiQdeletion (MCM2732) finishes with a higher cell performance index thanthe control cells that are wild type for yfiQ. The 64 hr points wereused to populate Table 3-3 above. CPI was calculated using the followingformula:CPI=total grams Isoprene/total grams dry cell weight

Smoothed specific isoprene productivity achieved in each 15-Lfermentation over time is shown in FIG. 8 . The experimental cellscarrying the yfiQ deletion (MCM2732) shows a higher peak specificproductivity than the control cells that are wild type for yfiQ.Presumably this is driven by the higher specific glucose uptake rate.The 64 hr points were used to populate Table 3-3 above. Smoothedspecific isoprene productivity was calculated using the followingformula:Specific productivity (mg/L/hr/OD)=IspER*68.117 g/mol/OD. IspER is theisoprene Evolution Rate in (mmol/L/hr). OD=optical density=Absorbance at550 nm*dilution factor in water.Smoothed Specific productivity (mg/L/hr/OD)=slope of milligrams isopreneproduced per hour(averaged over 8 hour interval)/broth volume*OD

The results of these assays suggest that the yfiQ deletion results in astrain (MCM2732) that does not accumulate acetate in the broth duringthis isoprene production process (FIG. 3 ). MCM2732 kept a consistentlylow broth concentration of acetate throughout the fermentation run.Presumably, and without being bound to theory, this is because the geneencoding yfiQ has been deleted leading to decreased acetylation ofacetyl-CoA synthetase. Decreased acetylation of acetyl-CoA synthetaseremains active and free to take up acetate from the broth. In contrast,control cells MCS1227 accumulated about 6 g/L of acetate and this istypical of what occurs is isoprene producing cells carrying a deletionfor ackA. Presumably, and without being bound to theory, this is due tothe fact that once acetate gets out of the cell, it cannot again betaken up by acetate kinase, since the gene encoding this polypeptide hasbeen deleted. In conclusion, the yfiQ deletion (MCM2732 stain) resultsin a higher specific glucose uptake rate (FIG. 4 ) which in turn has anumber of beneficial effects on isoprene performance (FIG. 5 ).

Example 4: Effect of yfiQ Deletion on Growth Rate and IsopreneProduction in Cells Grown with Acetate

The example explores the effect of deletion of yfiQ on cellular growthrate and isoprene production when cells are cultured in the presence ofacetate.

I. Materials and Methods

LB media, TM3 media without yeast extract and MgSO₄, 10% yeast extract,1M MgSO₄, 50% glucose, 200 mM IPTG, 50 mg/mL spectinomycin, 50 mg/mLcarbenicillin, 10% sulfuric acid and 100 mM Tris, 100 mM NaCl pH 7.6buffer were prepared in-house. Aluminum foil seal, 48-well sterile 5 mLblock, Breathe Easier sealing membrane, 96-well micro titer plates werepurchased from VWR. 96-well glass block was purchased from ZinsserAnalytical. Sodium acetate was purchased from Sigma. Agilent 6890 GC wasequipped with a 5973N Mass spectrometer. A summary of theisoprene-producing cells used in the example is in Table 4-1.

TABLE 4-1 Summary of Isoprene-Producing Cells Strain Name GenotypeMCM2732 MD891 + FRT-kan-FRT::YfiQ FRT-cmp-FRT::PL.2-pta pMCS1019pMCM1225 MCS1316 MD891 + FRT::PL.2-pta pMCS1019 pMCM1225

Overnight cultures were prepared directly from glycerol culture stocksin 3 mL of LB media with appropriate antibiotics in 10 mL plastic testtubes. Overnight cultures were grown at 30° C., 220 rpm.

Supplemented TM3 media was prepared by combining TM media, (withoutMgSO₄ and yeast extract) 1% glucose or various concentrations of sodiumacetate, 8 mM MgSO₄, 0.02% yeast extract and appropriate antibiotics. 2mL of day cultures were prepared in 48-well sterile block by inoculatingovernight culture in supplemented TM3 media at 0.2 optical density (OD).Blocks were sealed with Breathe Easier membranes and incubated for 2hours at 34° C., 600 rpm. After 2 hours of growth, OD was measured at600 nm in the micro-titer plate and cells were induced with 200 μM ofIPTG. OD reading and isoprene specific productivity samples were takenfrom 2-6 hours post induction. OD measurement was done in themicro-titer plate at appropriate dilution in the TM3 media at 600 nmusing a SpectraMax Plus190 (Molecular Devices).

100 μl of isoprene samples were collected in a 96 well glass block everyhour after IPTG induction for 4 hours. Glass block was sealed withaluminum foil and incubated at 34° C. while shaking at 450 rpm, for 30minutes on the thermomixer. After 30 minutes, the block was kept at 70°C. water bath for 2 minutes and isoprene headspace measurement was donein GC/MS.

II. Results

The growth rates of wild type and delta yfiQ isoprene producing cellsgrown on various concentrations of acetate as a sole carbon source areshown in FIG. 9 while isoprene specific productivity is shown in FIG. 10. In contrast, growth rates of wild type and delta yfiQ isopreneproducing cells grown using glucose as a sole carbon source are shown inFIG. 11 while isoprene specific productivity is shown in FIG. 12 forthis set of conditions.

Example 5: Effects of yfiQ Gene Deletion on Isoprenoid Production inCells

This example is performed to evaluate isoprenoid production usingmodified E. coli cells which express introduced genes from themevalonate pathway and farnesyl pyrophosphate (FPP) synthase (e.g.farnesene synthase codon-optimized for E. coli (SEQ ID NO:26) oramorphadiene synthase codon-optimized for E. coli (SEQ ID NO:25));geranyl pyrophosphate synthase; or geranylgeranyl pyrophosphatesynthase; and are grown in fed-batch culture at the 15-L scale. The celllines in this experiment carry a deletion in the gene encoding theacetate kinase (AckA) polypeptide and express an M. hominisphosphoketolase. Additionally, both over express phosphotransacetylase(pta).

These isoprenoid producing cells are run in the same process. Theperformance metrics of a control cells are compared to experimentalcells that has been deleted for yfiQ, a lysine acetyltransferase or haveincreased expression of cobB, a deacetylase. The relevant performancemetrics are cumulative isoprenoid yield on glucose, volumetricproductivity of isoprenoid and cell performance index.

I. Construction of Isoprenoid Producing Cells

Using standard techniques, an farnesyl pyrophosphate (FPP) synthase(e.g. farnesene synthase codon-optimized for E. coli (SEQ ID NO:26) oramorphadiene synthase codon-optimized for E. coli (SEQ ID NO:25));geranyl pyrophosphate synthase; or geranylgeranyl pyrophosphate synthasegene is cloned in place of ispS in either pMCS1019 or pEWL1421. Theresulting plasmid is co-transformed with pMCM1225 into a host strain. Apartial list of host strains are described in Table 5.

TABLE 5 Summary of Host Strains Host Strain Genotype MCM2721 MD891 +YfiQ::FRT-kan-FRT MCM2722 MCM2721 with kan looped out MCM2736 MD891 +CobB::FRT-kan-FRT MCM2740 MD891 + FRT-kan-FRT-gi1.6-YfiQ MCM2742 MD891 +FRT-kan-FRT-gi1.6-CobB (BL21 ATG) MCM2754 MCM2736 with kan looped outMCM2760 MCM2740 with kan looped out MCM2764 MCM2742 with kan looped outMCM2801 CTO pgl- FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT-kan-FRTclone A MCM2804 CTO pgl- FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRTclone 1 MCM3083 CTO pgl- FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRTpta::FRT-kanR-FRT clone A MCM3139 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT-kanR-FRT clone AFRT-cmp5-FRT::gi1.6-acs MD1243 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT clone A,FRT::gi1.6-acs, i actP::ML DW1242 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT clone A,FRT::gi1.6-acs, i gi1.6ackA MD1280 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, iyfiQ::Gi1.2-rpe_tktA_rpiA_talB ML MD1281 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, ipfkA_tag T ML, i yfiQ::Gi1.2-rpe_tktA_rpiA_talB ML MD1282 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, ipfkA_tag I ML, i yfiQ::Gi1.2-rpe_tktA_rpiA_talB ML MD1283 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, ipfkA_tag R ML, i yfiQ::Gi1.2-rpe_tktA_rpiA_talB ML

II. Materials and Methods

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 50% sulfuric acid 1.6 mL,1000× Modified Trace Metal Solution 1 mL. All of the components areadded together and dissolved in Di H₂O. This solution is heat sterilized(123° C. for 20 minutes). The pH is adjusted to 7.0 with ammoniumhydroxide (28%) and q.s. to volume. Glucose 10 g, Vitamin Solution 8 mL,and antibiotics are added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution (Per Liter):

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl, 10 g, FeSO₄*7H₂O, 1 g,CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O, 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O,pH are adjusted to 3.0 with HCl/NaOH, and then the solution is q.s. tovolume and filter sterilized with a 0.22 micron filter.

Vitamin Solution (Per Liter):

Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic acid 1.0 g,pyridoxine hydrochloride 4.0 g. Each component is dissolved one at atime in Di H₂O, pH are adjusted to 3.0 with HCl/NaOH, and then thesolution is q.s. to volume and filter sterilized with 0.22 micronfilter.

Macro Salt Solution (Per Liter):

MgSO₄*7H₂O 296 g, citric acid monohydrate 296 g, ferric ammonium citrate49.6 g. All components are dissolved in water, q.s. to volume and filtersterilized with 0.22 micron filter.

Feed Solution (Per Kilogram):

Glucose 0.590 kg, Di H₂O 0.393 kg, K₂HPO₄ 7.4 g, and 100% Foamblast8828.9 g. All components are mixed together and autoclaved. Afterautoclaving the feed solution, nutrient supplements are added to thefeed bottle in a sterile hood. Post sterilization additions to the feedare (per kilogram of feed solution), Macro Salt Solution 5.54 mL,Vitamin Solution 6.55 mL, 1000× Modified Trace Metal Solution 0.82 mL.For a target of 100 μM IPTG: 1.87 mL of a sterile 10 mg/mL solution isadded per kilogram of feed.

This experiment is carried out to monitor isoprenoid production fromglucose at the desired fermentation pH (7.0) and temperature (34° C.).To start each experiment, the appropriate frozen vial of the E. coliisoprenoid producing cells is thawed and inoculated into a flask withtryptone-yeast extract (LB) medium and the appropriate antibiotics.After growing to an optical density of approximately 1.0, measured at550 nm (OD₅₅₀), 500 mL is used to inoculate a 15-L bioreactor and bringthe initial tank volume to 5 L.

The inlet gas using to maintain bioreactor backpressure at 0.7 bar gaugeand to provide the oxygen to the cells is supplied by in housefacilities that dilute the inlet gas to a known concentration (7.3 to8.3 vol % oxygen).

The batched media has glucose batched in at 9.7 g/L. Induction isachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). Asyringe containing a sterile solution of IPTG is added to bring the IPTGconcentration to 100 μM when the cells are at an OD₅₅₀ of 6. Once theglucose is consumed by the culture, as signaled by a rise in pH, theglucose feed solution is fed to meet metabolic demands at rates lessthan or equal to 10 g/min. At a fixed time after dissolved oxygenlimitation is established, the temperature is raised from 34° C. to 37°C. over the course of one hour. The fermentation is run long enough todetermine the maximum cumulative isoprenoid mass yield on glucose,typically a total of 64 hrs elapsed fermentation time (EFT).

Isoprenoid, Oxygen, Nitrogen, and Carbon Dioxide levels in the off-gasare determined independently by a Hiden HPR20 (Hiden Analytical) massspectrometer. Dissolved Oxygen in the fermentation broth is measured bysanitary, sterilizable probe with an optical sensor provided HamiltonCompany. The citrate, glucose, acetate, and mevalonate concentrations inthe fermenter broth is determined in broth samples taken at 4 hourintervals by an HPLC analysis. Concentration in broth samples aredetermined by comparison of the refractive index response versus apreviously generated calibration curve using standard of a knownconcentration.

HPLC information is as follows: System: Waters Alliance 2695; Column:BioRad-Aminex HPX-87H Ion Exclusion Column 300 mm×7.8 mm Catalog#125-0140; Column Temperature: 50° C.; Guard column: BioRad—MicroguardCation H refill 30 mm×4.6 mm Catalog #125-0129; Running buffer: 0.01NH₂SO₄; Running buffer flow rate: 0.6 mL/min; Approximate runningpressure: ˜1100-1200 psi; Injection volume: 20 microliters; Detector:Refractive Index (Knauer K-2301;) Runtime: 26 minutes.

Example 6: Construction of Acetate Cycling Strains

This Example describes the construction of strains containing additionalmutations in genes responsible for the modulation of acetate cycling,acetate production, and acetyl-CoA production. This examples describesthe construction of strains carrying a deletion of phosphotransacetylase(pta) and a deletion of the yfiQ gene.

I. Construction of CMP400

A chloramphenicol resistance-marked constitutive gi1.6 promoter wasinserted in front of the acs gene using GeneBridges protocols. TheFRT-gb2-cm-FRT cassette was PCR amplified from plasmid supplied byGeneBridges using primers acsAUppKD3 and acsADnGI1.6pKD3R. The resultingPCR product, FRT-cmp-FRT::gi.6-acs, was transformed into strain HMBcarrying the pRedET plasmid following the GeneBridges protocol andrecombinants were selected at 37° C. on LB cmp5 plates. The HMB genotypeis: BL21 wt, pgl+t PL.2-mKKDyI::FRT. A colony was confirmed to be cmpRand carbS and then frozen as CMP400.

TABLE 6-1 Primer Sequences acsAUppKD3tcacgacagtaaccgcacctacactgtcatgacattgctcgcccctatgtgt SEQ ID NO: 59aacaaataaccacactgcccatggtccatatgaatatcctcc acsADnGI1.caacggtctgcgatgttggcaggaatggtgtgtttgtgaatttggctcatat SEQ ID NO: 606pKD3R ataattcctcctgctatttgttagtgaataaaagtggttgaattatttgctcaggatgtggcattgtcaagggcgtgtaggctggagctgcttcg

II. Construction of MD803

A 4.429 kb PCR fragment, Pta::Kan, was amplified from Keio Collectionusing primers CMP534 and CMP535. Approximately ˜300 ng of this PCRproduct was used to integrate into the host strain CMP1141 (HMBGi1.2gltA yhfSFRTPyddVIspAyhfS thiFRTtruncIspA pgl ML(pgl(−))+pRedETAmp). The transformants were selected at 37° C. onLA+Kan10 plates. The mutants were later verified with the same set ofprimers. The resulting strain was named MD803 (CMP1141+i pta::Kan).

TABLE 6-2 Primer Sequences CMP534 ackACF gtgcaaattcacaactcagcgg SEQ IDNO: 61 CMP535 PtaCR caccaacgtatcgggcattgc SEQ ID NO: 62

III. Construction of MCM3151

Strain MCM2065 (BL21, Δpgl PL.2mKKDyl, GI1.2gltA, yhfSFRTPyddVIspAyhfS,thiFRTtruncIspA, bMVK) was transduced with a P1 lysate of MCM2722(Example 1) using standard methods, with yfiQ::kanR transductantsselected on LB kan10 plates at 37° C. overnight. Transductants wererestreaked and confirmed by PCR. This strain was grown in LB kan10 andfrozen as MCM2801. The kanR marker was looped out by transformation ofplasmid pCP20 (see worldwide webcgsc.biology.yale.edu/Site.php?ID=64621), selection on LB carb50 at 30°C. overnight followed by passage of a single colony at 37° C. in liquidLB carb50 until visibly turbid. Culture was streaked on LB withoutantibiotics and grown at 37° C. overnight. Single colonies were patchedto plates with and without antibiotics and a carbS, kanS colony wasidentified. PCR was used to confirm the presence of a wildtype ackAlocus and an unmarked ΔyfiQ locus. This colony was grown in LB at 37° C.and frozen as MCM2804. A P1 lysate from MD803 was used to transduceMCM2804 with pta::FRT-kan-FRT, with transductants selected on LB kan10plates at 37° C. overnight. A colony was restreaked on LB kan10, grownin liquid broth and frozen as MCM3803. A P1 lysate grown on MCS1388, asubclone of CMP400, was used to transduce MCM3803 withFRT-cmp-FRT::gi.6-acs. Transductants were selected on LB cmp5 plates at37° C. overnight. A colony was streaked on LB cmp5, grown overnight, andthen a resulting single colony was used to inoculate a liquid culture.This culture was frozen as MCM3139. Plasmids pMCM1225 and pMCS1019 wereco-electroporated into MCM3139 and transformants selected on a LB carb50spec50 plate incubated at 37° C. overnight. A single colony was grown inliquid LB carb50 spec50 at 37° C. and frozen as MCM3151.

TABLE 6-3 Descriptions of Cells MCM # Genotype Parent MCM2801 CTO pgl-FRT-PL.2-2cis-RBS10000- MCM2065 MVK(burtonii) yfiQ::FRT-kan-FRT clone AMCM2804 CTO pgl- FRT-PL.2-2cis-RBS10000- MCM2801 MVK(burtonii) yfiQ::FRTclone 1 MCM3083 CTO pgl- FRT-PL.2-2cis-RBS10000- MCM2804 MVK(burtonii)yfiQ::FRT pta::FRT-kanR- FRT clone A MCM3139 CTO pgl-FRT-PL.2-2cis-RBS10000- MCM3083 MVK(burtonii) yfiQ::FRT pta::FRT-kanR-FRT clone A FRT-cmp5-FRT::gi1.6-acs MCM3151 CTO pgl-FRT-PL.2-2cis-RBS10000- MCM3139 MVK(burtonii) yfiQ::FRT pta::FRT-kanR-FRT clone A FRT-cmp5-FRT::gi1.6-acs + pMCS1019 + pMCM1225

IV. Construction of Isoprene Producing Cells MD1206 (M. hominisPhosphoketolase) and MD1207 (E. Gal Phosphoketolase)

MCM3139 was inoculated and grown overnight. The culture was diluted andgrown to OD 0.8-1.0, then were washed and electroporated with plasmidpCP20 (see worldwide web cgsc.biology.yale.edu/Site.php?ID=64621). Theculture was recovered for 1 hour at 30° C. Transformants were selectedon LA+Carb50 plates and incubated overnight at 30° C. The resulting CmRmarker-less strain was named MD1205 (CTOpgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT clone A,FRT::gi1.6-acs). MD1205 was co-transformed with pMCM1225 and pMCS1019 togenerate MD1206 or pMCM1225 and pEWL1421 to generate MD1207.Transformants were selected on LA+Spec50+Carb50 plates.

TABLE 6-4 Descriptions of Cells MCM3139 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT-kanR-FRT clone AFRT-cmp5-FRT::gi1.6-acs MD1205 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT clone A,FRT::gi1.6-acs MD1206 CTO pgl- FRT-PL.2-2cis-RBS10000-MVK(burtonii)yfiQ::FRT pta::FRT clone A, FRT::gi1.6-acs + pMCM1225 + pMCS1019 MD1207CTO pgl- FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT cloneA, FRT::gi1.6-acs + pMCM1225 + pEWL1421

Example 7: Effects of Example 6 Strains on Isoprene Yield

This example measures isoprene production in cells carrying a deletionof phosphotransacetylase (pta) and a deletion of the yfiQ gene. Celldetails are provided in Table 7-1.

Strain MCM2732 with the deletion of yfiQ and acs constitutively activedisplayed improved acetate reuptake and isoprene production. Theimproved reuptake of acetate significantly lowered acetate accumulationassociated with ackA minus strains, as well as increased isoprenespecific productivity, and improved viability, extending the productiveportion of the run, which increased isoprene titer. This example usesMCM3151, MD1206, and MD1207 which enhance the acetate reuptake bydeleting pta and thereby increasing phosphoketolase flux (AcP toisoprene) (FIG. 28 ).

TABLE 7-1 Cells Used in the Example Upper Isoprene synthase/ pathwayPhosphoketolase Host plasmid plasmid MD1207 CTO pgl IPTG inducible PtrcIPTG inducible bLP expressing (pTrc IspS E. gal PKL) GI1.6acsA E.gallinarum mvaE, (Carb 50) pta- mvaS (pMCM1225 Spec 50) MD1206 CTO pglIPTG inducible Ptrc IPTG inducible bLP expressing (pTrc IspS M. hominisPKL) GI1.6acsA E. gallinarum mvaE, (Carb 50) pta- mvaS (pMCM1225 Spec50) MCM3151 CTO pgl IPTG inducible Ptrc IPTG inducible bLP expressing(pTrc IspS M. hominis PKL) GI1.6acsA::Chlor E. gallinarum mvaE, (Carb50) pta::Kan mvaS (pMCM1225 Spec 50) MCM2732 MD891 + IPTG inducible PtrcIPTG inducible (Control FRT-kan- expressing (pTrc_IspS_RBS3_PKL16 cells)FRT::yfiQ E. gallinarum mvaE, [M. hominis]) FRT-cmp- mvaS (pMCM1225(pMCS1019, Carb 50) FRT::PL.2-pta Spec 50)

I. Materials and Methods

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 50% sulfuric acid 1.6 mL,1000× Modified Trace Metal Solution 1 ml. All of the components wereadded together and dissolved in Di H₂O. This solution was heatsterilized (123° C. for 20 minutes). The pH was adjusted to 7.0 withammonium hydroxide (28%) and q.s. to volume. Glucose 10 g, VitaminSolution 8 mL, and antibiotics were added after sterilization and pHadjustment.

1000× Modified Trace Metal Solution (Per Liter):

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in DiH₂O, pH was adjusted to 3.0 with HCl/NaOH, and then the solution wasq.s. to volume and filter sterilized with a 0.22 micron filter.

Vitamin Solution (Per Liter):

Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic acid 1.0 g,pyridoxine hydrochloride 4.0 g. Each component was dissolved one at atime in Di H₂O, pH was adjusted to 3.0 with HCl/NaOH, and then thesolution was q.s. to volume and filter sterilized with 0.22 micronfilter.

Macro Salt Solution (Per Liter):

MgSO₄*7H₂O 296 g, citric acid monohydrate 296 g, ferric ammonium citrate49.6 g. All components were dissolved in water, q.s. to volume andfilter sterilized with 0.22 micron filter.

Feed Solution (Per Kilogram):

Glucose 0.590 kg, Di H₂O 0.393 kg, K₂HPO₄ 7.4 g, and 100% Foamblast8828.9 g. All components were mixed together and autoclaved. Afterautoclaving the feed solution, nutrient supplements are added to thefeed bottle in a sterile hood. Post sterilization additions to the feedare (per kilogram of feed solution), Macro Salt Solution 5.54 ml,Vitamin Solution 6.55 ml, 1000× Modified Trace Metal Solution 0.82 ml.For a target of 100 μM IPTG: 1.87 ml of a sterile 10 mg/ml solution isadded per kilogram of feed.

This experiment was carried out to monitor isoprene production fromglucose at the desired fermentation pH (7.0) and temperature (34° C.).To start each experiment, the appropriate frozen vial of the E. coliisoprene producing cells was thawed and inoculated into a flask withtryptone-yeast extract (LB) medium and the appropriate antibiotics.After the inoculum grew to an optical density of approximately 1.0,measured at 550 nm (OD₅₅₀), 500 mL was used to inoculate a 15-Lbioreactor and bring the initial tank volume to 5 L.

The inlet gas using to maintain bioreactor backpressure at 0.7 bar gaugeand to provide the oxygen to the cells was supplied by in housefacilities that dilute the inlet gas to a known concentration (7.3 to8.3 vol % oxygen).

The batched media had glucose batched in at 9.7 g/L. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). Asyringe containing a sterile solution of IPTG was added to bring theIPTG concentration to 100 μM when the cells were at an OD₅₅₀ of 6. Oncethe glucose was consumed by the culture, as signaled by a rise in pH,the glucose feed solution was fed to meet metabolic demands at ratesless than or equal to 10 g/min. At a fixed time after dissolved oxygenlimitation was established, the temperature was raised from 34° C. to37° C. over the course of one hour. The fermentation was run long enoughto determine the maximum cumulative isoprene mass yield on glucose,typically a total of 64 hrs elapsed fermentation time (EFT). Table 7-2shows process conditions.

TABLE 7-2 Process Conditions Target [IPTG] after Batched bolus additionTarget [IPTG] Strain [IPTG] at ~EFT 6-8 hrs in Feed Bottle Used (μM)(μM) (μM) MCS922 1.4 100 100 MCM2732 1.4 100 100

Isoprene, Oxygen, Nitrogen, and Carbon Dioxide levels in the off-gaswere determined independently by a Hiden HPR20 (Hiden Analytical) massspectrometer.

Dissolved Oxygen in the fermentation broth is measured by sanitary,sterilizable probe with an optical sensor provided Hamilton Company.

The citrate, glucose, acetate, and mevalonate concentrations in thefermenter broth was determined in broth samples taken at 4 hourintervals by an HPLC analysis. Concentration in broth samples weredetermined by comparison of the refractive index response versus apreviously generated calibration curve using standard of a knownconcentration.

HPLC Information was as follows: System: Waters Alliance 2695; Column:BioRad-Aminex HPX-87H Ion Exclusion Column 300 mm×7.8 mm Catalog#125-0140; Column Temperature: 50° C.; Guard column: BioRad—MicroguardCation H refill 30 mm×4.6 mm Catalog #125-0129; Running buffer: 0.01NH₂SO₄; Running buffer flow rate: 0.6 ml/min; Approximate runningpressure: ˜1100-1200 psi; Injection volume: 20 microliters; Detector:Refractive Index (Knauer K-2301); Runtime: 26 minutes

II. Results

Isoprene Productivity Metrics (and EFT when the value was taken) areshown in Table 7-3.

Acetate concentration was kept low, even though pta was deleted in theexperimental strains, so it would appear that acs overexpression,coupled with yfiQ deletion was sufficient to route the acetyl phosphate(from the phosphoketolase pathway) to acetyl-CoA (and onto themevalonate pathway, producing isoprene).

The MD1207 strain did not perform as well as the other new strains. Itis interesting that this strain is expressing the E. gallinarumphosphoketolase which typically shows a higher expression level comparedto the M. hominis phosphoketolase. The increased lag after inductionresulted in a slower growth rate, and longer time to reach DO %limitation, thereby shortening the peak productivity and the highyielding phases of the run.

TABLE 7-3 Isoprene Productivity CPI Overall (Total g Isoprene Isopreneisoprene/ Titer Volumetric total Peak (gram Cumulative ProductivitygDCW) at Peak Instantaneous isoprene/ % Yield of at time of Max time ofmax Specific yield of average isoprene on max overall Optical overallProductivity isoprene volume of glucose isoprene Density isoprene (mgisoprene/ on glucose tank broth Strain Name (g/g %) yield (g/L/hr)(A550) yield L/hr/OD) (g/g %) in Liters) MD1207 19.40 2.04 106.7  3.3643.1 24.5 122.5 (60 hrs) (60 hrs) (32 hrs) (60 hrs) (32 hrs) (52.4hrs)   (60 hrs) MD1206 20.45 2.55 93.9 3.27 53.4 24.0 153.1 (60 hrs) (60hrs) (28 hrs) (60 hrs) (28 hrs) (48 hrs) (60 hrs) MCM3151 19.24 2.3796.6 2.73 50.1 22.8 142.3 (60 hrs) (60 hrs) (36 hrs) (60 hrs) (28 hrs)(40 hrs) (60 hrs) MCM2732 19.64 2.34 99.6 3.53 47.0 23.8 140.3 (60 hrs)(60 hrs) (24 hrs) (60 hrs) (28 hrs) (48 hrs) (60 hrs)

Broth concentration of acetate measured in each 15-L fermentation overtime is shown in FIG. 16 . In all cases, control and experimental, thebroth concentration of acetate was very low. The experimental strainshad a slightly lower acetate concentration at the end of fermentation.

Cumulative yield of isoprene on glucose achieved in each 15-Lfermentation over time is shown in FIG. 17 . The experimental cells thatare deleted for pta (MD1206) finishes with a higher cumulative yield ofisoprene on glucose than the control cells that overexpresses pta(MCM2732). The 60 hr points were used to populate Table 7-3 above.Overall yield was calculated using the following formula:% wt Yield on glucose=Isoprene total(t)/[(Feed Wt(0)−FeedWt(t)+83.5)*0.59)],where 0.59 is the wt % of glucose in the glucose feed solution and 83.5is the grams of this feed batched into the fermenter at t=0. Each feedhad its weight % measured independently.

Yield of Isoprene on glucose (over previous 40 hr period) achieved ineach 15-L fermentation over time is shown in FIG. 18 . The experimentalcells that is deleted for pta (MD1206) finishes with a higher peak “40hr” yield of isoprene on glucose than the control cells thatoverexpresses pta (MCM2732). “40 hr” yield was calculated using thefollowing formula:% wt Yield on glucose=Isoprene total(t _(initial) −t ⁻⁴⁰)/[(Feed Wt(t_(initial) −t ⁻⁴⁰)*0.59)],where 0.59 is the wt % of glucose in the glucose feed solution. Eachfeed had its weight % measured independently.

Example 8: Construction of Additional Acetate Cycling Strains

This Example describes the construction of additional strains containingmutations in genes responsible for acetate production, acetate cycling,and acetyl-CoA production. The ackA gene was overexpressed to drive theconversion of acetate to acetyl-CoA. In another strain, actP was deletedto minimize transport of acetate across the membrane. Without beingbound to theory, it is believed that if acetate production is coupledwith transport across the membrane, this could result in energy loss dueto decoupling of the proton gradient.

The construct for overexpression of ackA was constructed using standardmolecular biology techniques (SEQ ID NO:109). Briefly, flanking regionsof ackA were fused to a heterologous promoter (GI1.2) in an allelicexchange cassette by seamless cloning and assembly (Life Technologies).The actP deletion construct was also generated by fusing homologousflanking regions to the allelic exchange cassette (SEQ ID NO:110). TheackA and actP vectors (FIG. 19 ) were isolated and then transformed intoMD1205 to generate DW1242 and MD1243 respectively. Positive integrantswere selected for resistance to tetracycline and markerless deletionstrains were identified by passaging on 5% sucrose. Final mutant strainswere confirmed by PCR and sequencing. Co-transformation of pMCM1225 andpEWL1421 into DW1242 generated DW1245 and into MD1243 generated MD1245.

TABLE 8-1 Primer Sequences Primer Name Sequence SEQ ID NO TS Fortcctaatttagttgacactctatcattg SEQ ID NO: 63 TS Revccatcttgttgagaaataaaagaaaatgcca SEQ ID NO: 64 actP Up Fortttatttctcaacaagatgggcaggctatcgcgatgccatcgtaac SEQ ID NO: 65 actP Up Revggagagattacatgatgcttgtacctcatgcagga SEQ ID NO: 66 actP Down Foraagcatcatgtaatctctccccttccccggtcgcctga SEQ ID NO: 67 actP Down Revagtgtcaacaaaaattaggacgtaaccaccatttactgtctgtgga SEQ ID NO: 68actP Test For ctggcgtagtcgagaagctgcttga SEQ ID NO: 69 actP Test Revgcatagcggaacatgaatttagagt SEQ ID NO: 70 ackA Up Fortttatttctcaacaagatggcggatcgagcatagtcatcatcttgtact SEQ ID NO: 71ackA Up GI cggttgatttgtttagtggttgaattatttgctcaggatgtggcatngtcaaggSEQ ID NO: 72 Rev gcgaatttgacgactcaatgaatatgtact ackA Down GIaccactaaacaaatcaaccgcgtttcccggaggtaacctaaaggaggtaaa SEQ ID NO: 73 Foraaaacatgtcgagtaagttagtactggttctga ackA Down Revagtgtcaacaaaaattaggagtacccatgaccagaccttccagc SEQ ID NO: 74ackA Up PL Rev atcaccgccagtggtatttangtcaacaccgccagagataatttatcaccgcSEQ ID NO: 75 agatggttatctgaatttgacgactcaatgaatatgtact ackA Down PLtaaataccactggcggtgatactgagcacatcagcaggacgcactgcaaa SEQ ID NO: 76 Forggaggtaaaaaaacatgtcgagtaagttagtactggttctga ackA EX Testtgcaggcgacggtaacgttcagcat SEQ ID NO: 77 For ackA EX Testgtggaagatgatcgccggatcgata SEQ ID NO: 78 Rev R6K TS Revagtgtcaacaaaaattaggactgtcagccgttaagtgttcctgtgt SEQ ID NO: 79actP R6K For ggtggttacgcagttcaacctgttgatagtacgta SEQ ID NO: 80actP R6K Rev ggttgaactgcgtaaccaccatttactgtctgtgga SEQ ID NO: 81

TABLE 8-2 Strain Descriptions Strain Description MD1243 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT clone A,FRT::gi1.6-acs, i actP::ML MD1245 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT clone A,FRT::gi1.6-acs, i actP::ML + pMCM1225 + pEWL1421 DW1242 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT clone A,FRT::gi1.6-acs, i gi1.6ackA DW1245 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) yfiQ::FRT pta::FRT clone A,FRT::gi1.6-acs, i gi1.6ackA + pMCM1225 + pEWL1421

Example 9: Effect of Example 8 Strains on Isoprene Yield and Growth Rate

This example measured isoprene production and growth rate in cellscarrying a deletion of the yfiQ gene and either an actP deletion or anackA overexpression.

I. Materials and Methods

LB media, TM3 media without Yeast extract and MgSO₄, 10% Yeast extract,1M MgSO₄, 50% Glucose, 200 mM IPTG, 50 mg/mL Spectinomycin, 50 mg/mLCarbenicillin, Aluminum foil seal, 48-well sterile 5 mL block, breatheeasier sealing membrane, aluminum foil seal, 96-well micro titer plates,96-well glass block purchased from Zinsser Analytic. Agilent 6890 GCequipped with a 5973N Mass spectrometer.

Supplemented TM3 media was prepared by combining TM media, (withoutMgSO₄ and Yeast extract) 1% Glucose, 8 mM MgSO₄, 0.02% Yeast extract andappropriate antibiotics. 2 mL of day culture was started in 48-wellsterile block by inoculating overnight culture in supplemented TM3 mediaat 0.2 optical density (OD). Blocks were sealed with breathe easiermembrane and incubated for 2 hours at 34° C., 600 rpm. After 2 hours ofgrowth, OD was measured at 600 nm in the micro titer plate and cellswere induced with 200 μM IPTG. OD reading was taken every hour after theIPTG induction for 4 hours to determine growth rate. OD was measurementwas done in the micro titer plate at appropriate dilution in the TM3media at 600 nm using a SpectraMax Plus190 (Molecular Devices).

100 μL of isoprene samples were collected in a 96 well glass block at 2,3 and 4 hours after IPTG induction. Glass block was sealed with aluminumfoil and incubated at 34° C. while shaking at 450 rpm, for 30 minutes onthe thermomixer. After 30 minutes, the block was kept in 70° C. waterbath for 2 minutes and isoprene headspace measurement was done in GC/MSto determine specific productivity.

II. Results

Both the actP deletion (MD1245) and the ackA overexpression (DW1245)cells displayed higher carbon dioxide evolution rates (CER), indicatingimproved respiration rates compared to the control cells (FIG. 20A-D).Both cells displayed improved isoprene titer and specific productivityas compared to the control, and the actP cells displayed an improvementin isoprene yield (FIG. 21A-D). These results show that improvementsaround acetate production and/or acetate cycling have a beneficialeffect on several different parameters of isoprene production. Withoutbeing bound by theory, it is possible that this effect is achieved byoptimizing the fluxes through glycolysis and phosphoketolase.

Example 10: Construction of Pentose Phosphate Pathway Modulation Strains

This Example describes the construction of strains containing mutationsin genes responsible for modulating the Pentose Phosphate Pathway (PPP).

Without being bound by theory, it is believed that the four genes in thepentose phosphate pathway critical for balancing carbon flux in aphosphoketolase-expressing host are tktA, talB, rpe, and rpiA.Maximizing the cycling of carbon towards X5P could optimize the splitbetween fluxes through glycolysis, the pentose phosphate pathway, andphosphoketolase. The construct described below was designed tooverexpress and integrate all four non-oxidative pentose phosphate genesin the yfiQ locus in the chromosome. This construct represents only oneattempt at improving the routing of carbon through PPP, and it is verylikely that refinement of this construct, by the addition of morepromoters, terminators, rearranging the genes, etc., will help todetermine what is the optimum expression level to balance flux throughphosphoketolase.

In addition to four genes of the PPP discussed above, modulation of thepentose phosphate pathway can be achieved by modulating PfkA. Withoutbeing bound by theory, it is believed that PfkA controls a major entrypoint and regulated node in glycolysis, and it is likely that forphosphoketolase (PKL) to function properly, phosphofructokinase activitymust be decreased. This would increase available fructose 6-phosphate(F6P) and drive carbon flux through pentose phosphate towards xylulose5-phosphate (X5P), the other substrate of phosphoketolase.

I. Construction of tktA, talB, rpe, and rpiA Mutant Strains

PPP genes were optimized and ordered from IDT as gBlocks double strandedgene fragments. All four genes were TOPO cloned into the pCR2.1 vector(Life Technologies) and sequenced prior to subsequent cloning. Initialvectors were built by seamless assembly (Life Technologies) with eitherthe GI1.2 or GI1.6 promoters and flanking sites for homologousrecombination. The final vector with all four PPP genes in a singleoperon were also built by seamless cloning (Life Technologies) (FIG. 22) (SEQ ID NO:111). This plasmid was transformed into MD1205 for allelicexchange, and strains were isolated by resistance to tetracycline.Independent strains were selected by resistance to 5% sucrose, and thepresence of the insertion in the proper genomic locus was verified byPCR. This cell was frozen as MD1280. MD1280 was co-transfected withpMCM1225 and pEWL1421 to generate MD1284.

TABLE 10-1 Primers Sequences Primer Name sequence SEQ ID NOyfiQ DOWN For tttatttctcaacaagatggggccgattaacatcatccagacgatSEQ ID NO: 82 yfiQ DOWN cggttgatttgtttagtggttgaattatttgctcaggatgtggcaSEQ ID NO: 83 GI1.6 Rev ttgtcaagggctcttgcccaacgcgaggaatcatgagtayfiQ DOWN cggttgatttgtttagtggttgaattatttgctcaggatgtggca SEQ ID NO: 84GI1.2 Rev tcgtcaagggctcttgcccaacgcgaggaatcatgagta yfiQ UP GIaccactaaacaaatcaaccgcgtttcccggaggtaacctaaagga SEQ ID NO: 85 Forggtaaaaaaacaccggtctcccgcagaagtgaccga yfiQ UP Revactatcaacaggttgaactgcgccgttcgatagctggctgaacga SEQ ID NO: 86yfiQ Test For gcatcacgcagctcctggcggaaca SEQ ID NO: 87 yfiQ Test Revgctgaacgtgaattgagcagtcgct SEQ ID NO: 88 rpe R6K Fortacacacataaggaggttcccaatgaaacagtatctgatcgcacc SEQ ID NO: 89 tagcarpe R6K Rev tattcgaatgtatgctagtggacgtcaatcattactcgtggctca SEQ ID NO: 90ctttcgccagttca rpe R6K For cactagcatacattcgaataaggaggaatactatgtcatctcgtaSEQ ID NO: 91 aggaactggcgaa tkt R6K Revtatctccttcttgagccgattatcattacagcagctctttggctt SEQ ID NO: 92 tcgcgacarpi R6K For atcggctcaagaaggagatatacatatgacgcaggacgaactgaa SEQ ID NO: 93aaaagcggt rpi R6K Rev tattcctccttcaggacctttcattatttaacgatcgttttgacgSEQ ID NO: 94 ccatc tal R6K Foraaggtcctgaaggaggaataaaccatgaccgataaactgaccagc SEQ ID NO: 95 ctgcgttal R6K Rev gaccggttcattacagcaggtcgccgatcattttctcca SEQ ID NO: 96R6K Plasmid cctgctgtaatgaaccggtctcccgcagaagtgaccgaatga SEQ ID NO: 97 ForR6K Plasmid ggaacctccttatgtgtgtaaacctttaggttacctccgggaaac SEQ ID NO: 98Rev gcggttga

TABLE 10-2 Strain Descriptions Strain Description MD1280 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, iyfiQ::Gi1.2-rpe_tktA_rpiA_talB ML MD1281 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, ipfkA_tag T ML, i yfiQ::Gi1.2-rpe_tktA_rpiA_talB ML MD1282 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, ipfkA_Tag I ML, i yfiQ::Gi1.2-rpe_tktA_rpiA_talB ML MD1283 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, ipfkA_Tag R ML, i yfiQ::Gi1.2-rpe_tktA_rpiA_talB ML MD1284 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, iyfiQ::Gi1.2-rpe_tktA_rpiA_talB ML + pMCM1225 + pEWL1421 MD1285 CTO pgl-FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A, FRT::Gi1.6-acs, ipfkA_Tag T ML, i yfiQ::Gi1.2-rpe_tktA_rpiA_talB ML + pMCM1225 + pEWL1421MD1286 CTO pgl- FRT-PL.2-2cis-RBS10000-MVK(burtonii) pta::FRT clone A,FRT::Gi1.6-acs, i pfkA_Tag I ML, i yfiQ::Gi1.2-rpe_tktA_rpiA_talB ML +pMCM1225 + pEWL1421 MD1287 CTO pgl- FRT-PL.2-2cis-RBS10000-MVK(burtonii)pta::FRT clone A, FRT::Gi1.6-acs, i pfkA_Tag R ML, iyfiQ::Gi1.2-rpe_tktA_rpiA_talB ML + pMCM1225 + pEWL1421

II. Construction of pfkA Mutant Strains

Partially functional proteolytic tags were generated to downregulatepfkA. Random mutations at the third to last amino acid position in tmRNAwere fused to the C-terminus of PfkA using GeneBridges recombineering(following the manufacturer's recommended protocol) in a WT BL21background (FIG. 23 ). Mutants were then screened for growth in TM3glucose in the growth profiler (FIG. 24 ). The variant with isoleucine(I) in the 3^(rd) to last position is likely very similar to leucine(L), the native amino acid. This accordingly grew the slowest due to thehigh protein degradation rate of a “WT” tag. The variant with arginine(R) displayed a more modest effect, and threonine (T) was the leastimpaired. These three variants were selected and moved into MD1205 orMD1280 using the R6K allelic exchange method. Briefly, PCR fragmentsgenerated from primers listed below were used with tet-sac (TS) and R6Kfragments in seamless cloning reactions (Life Technologies) to yield theplasmids for allelic exchange (SEQ ID NO:112). These plasmids were thenintroduced into MD1205 or MD1280 by selection for tetracyclineresistance, and then counter selected by subsequent plating onto mediumcontaining 5% sucrose. Strains harboring the individual proteolytic tagmutations in pfkA were identified by PCR and sequencing. pMCM1225 andpEWL1421 transfected into each cell using standard molecular biologytechniques. See Table 10-2 for strain details.

TABLE 10-3 Primer Sequences Primer Name sequence SEQ ID NO pfKA tmRNAtgaagcgtccgttcaaaggcgactggctagactgcgcgaaaaaactgtat SEQ ID NO: 99 XAA Forgctgctaacgatgaaaattatgctnnngctgcataaaattaaccctcactaa agggcg pfkA tmRNAgcttctgtcatcggtttcaggctaaaggaatctgcctttttccgaaatcataat SEQ ID NO: 100Rev acgactcactatagggctc pfkA UP Fortttatttctcaacaagatgggttatcggcggtgacggttcctacat SEQ ID NO: 101pfkA UP Rev agcataattttcatcgttagcagcatacagttttttcgcgcagtctagccagtcSEQ ID NO: 102 gcct pfkA DOWN Rctaacgatgaaaattatgctcgcgctgcataatgatttcggaaaaaggcag SEQ ID NO: 103 Forattcct pfkA DOWN I ctaacgatgaaaattatgctattgctgcataatgatttcggaaaaaggcagaSEQ ID NO: 104 For ttcct pfkA DOWN Tctaacgatgaaaattatgctacggctgcataatgatttcggaaaaaggcag SEQ ID NO: 105 Forattcct pfkA DOWN actatcaacaggttgaactgcggtgcggagttatccggcagacgtSEQ ID NO: 106 Rev pfkA Test For ctgacatgatcaaccgtggcggta SEQ ID NO: 107pfkA Test Rev gatcgttccagtcatggatctgct SEQ ID NO: 108

Example 11: Effects of the Modulation of the Pentose Phosphate Pathwayon Isoprene Yield

I. Materials and Methods

LB media, TM3 media without Yeast extract and MgSO₄, 10% Yeast extract,1M MgSO₄, 50% Glucose, 200 mM IPTG, 50 mg/mL Spectinomycin, 50 mg/mLCarbenicillin, Aluminum foil seal, 48-well sterile 5 mL block, breatheeasier sealing membrane, aluminum foil seal, 96-well micro titer plates,96-well glass block purchased from Zinsser Analytic. Agilent 6890 GCequipped with a 5973N Mass spectrometer.

Supplemented TM3 media was prepared by combining TM media, (withoutMgSO₄ and Yeast extract) 1% Glucose, 8 mM MgSO₄, 0.02% Yeast extract andappropriate antibiotics. 2 mL of day culture was started in 48-wellsterile block by inoculating overnight culture in supplemented TM3 mediaat 0.2 optical density (OD). Blocks were sealed with breathe easiermembrane and incubated for 2 hours at 34° C., 600 rpm. After 2 hours ofgrowth, OD was measured at 600 nm in the micro titer plate and cellswere induced with 200 μM IPTG. OD reading was taken every hour after theIPTG induction for 4 hours to determine growth rate. OD was measurementwas done in the micro titer plate at appropriate dilution in the TM3media at 600 nm using a SpectraMax Plus190 (Molecular Devices).

100 μL of isoprene samples were collected in a 96 well glass block at 2,3 and 4 hours after IPTG induction. Glass block was sealed with aluminumfoil and incubated at 34° C. while shaking at 450 rpm, for 30 minutes onthe thermomixer. After 30 minutes, the block was kept in 70° C. waterbath for 2 minutes and isoprene headspace measurement was done in GC/MSto determine specific productivity.

II. Results

Cells with both the pentose phosphate pathway upregulated and pfkAdownregulated were assayed under fed batch conditions. The cells withonly the pentose phosphate pathway upregulated (MD1284), performed wellcompared to the control MD1207 cells (FIGS. 25A-D and 26A-D). The PPPupregulated cells displayed improved growth over the control, andadditionally displayed faster instantaneous isoprene/CO₂ production andhigher yield. Upregulation of the pentose phosphate pathway using acombination of the four non-oxidative genes is therefore beneficial forisoprene production. This could be due to an increase in availablesubstrate for PKL (xylulose-5-phosphate), or more effective balancing ofpentose phosphate intermediates (such as glyceraldehyde-3-phosphate orsedoheptulose-7-phosphate) that could benefit the overall flux throughthe pathway. All cells with pfkA downregulated by proteolytic tag grewpoorly and underperformed compared to the control cells (FIG. 27A-B).This was likely due to too high of a degradation rate for all threetags, despite the broad effect on growth in Example 10. Additionalmutations in pfkA tags would likely allow for improved growth rateswhile still driving the proper flux partition between glycolysis andphosphoketolase.

Example 12: Construction of Saccharomyces cerevisiae Reference andAcetylation Modulatory Strains

This example describes the construction of reference strains and strainscontaining mutations in genes responsible for modulating intracellularprotein acetylation. This example also describes measuring the effectsof an acetyltransferase gene deletion on isoprene production inSaccharomyces cerevisiae strains expressing the mevalonate pathway andisoprene synthase

I. Construction of Reference Strains with Constructs for Expression ofthe P. tremuloides Isoprene Synthase and Phosphoketolase Pathway inSaccharomyces cerevisiae

The Saccharomyces cerevisiae codon-optimized P. tremuloides isoprenesynthase and E. gallinarum phosphoketolase genes are synthesized by DNA2.0. Using molecular biology techniques, the genes are PCR amplified andcloned into a construct suitable for insertion into a Saccharomycescerevisiae 2 micron plasmid or chromosome. The construct has then thefollowing structure: Upperhomology-Promoter-Gene-Terminator-marker-Lower homology). The yeastcells are transformed and transformants are selected. Several coloniesare picked and inoculated into YPD medium (Yeast extract 10 g/L, Bactopeptone 20 g/L, glucose 20 g/L) and grown. DNA is isolated from thecultures and purified. Constructs are PCR-amplified with error-proof DNApolymerase and are sequenced to verify that the DNA sequence is correct.In addition, in some strains, one or more MVA pathway polypeptides aresimilarly expressed in the yeast host cells.

II. Construction of Reference Strains Containing a Deletion of aSaccharomyces cerevisiae Acetyltransferase Gene

Strains as described above containing also a deletion of a Saccharomycescerevisiae acetyltransferase are generated using standard molecularbiology protocols.

III. Production of Isoprene in Recombinant Strains of Saccharomycescerevisiae

One ml of 15 and 36 hour old cultures of isoprene synthase transformantsdescribed above are transferred to head space vials. The vials aresealed and incubated for 5 hours at 30° C. Head space gas is measuredand isoprene is identified by the methods described above.

Example 13: Construction of Saccharomyces cerevisiae AcetylationModulatory Strains that Further Contain Mutations in Genes Responsiblefor Either Modulating the Pentose Phosphate Pathway (PPP) or AcetateCycling

This Example describes the construction of yeast strains from Example 12further containing mutations in genes responsible for modulating thePentose Phosphate Pathway (PPP) or acetate cycling. This Example alsodescribes measuring the effects these mutations on isoprene production.

I. Construction of Reference Strains Containing Overexpression of One orMore Saccharomyces cerevisiae PPP Genes, or Expression or Overexpressionof Acetate Cycling Genes

Strains created in Example 12 above (containing mutated acetylationproteins, isoprene synthase, phosphoketolase and MVA pathwaypolypeptides) are further engineered to create strains containingmutations of Saccharomyces cerevisiae PPP genes. These are generatedusing standard molecular biology protocols. TKL1, TAL1, RPE1 and RKI1genes in the yeast pentose phosphate pathway involved in balancingcarbon flux in a phosphoketolase-expressing host are optimized forexpression in yeast. The genes are cloned and assembled to expressdifferent amount of the 4 activities of the PPP pathway using standardmolecular biology techniques. These plasmids are transformed into yeastcells, and strains are isolated, and frozen down. Independent strainsare verified by PCR. Alternatively, or additionally, strains created inExample 12 above are further engineered to contain mutations in theacetate cycling genes. One set of constructs expresses the E. coliphosphotransacetylase (codon-optimized for Saccharomyces cerevisiae andsynthesized by DNA2.0), and yet another series expresses E. coli acetatekinase (codon-optimized for Saccharomyces cerevisiae and synthesized byDNA2.0) with or without overexpression of Saccharomyces cerevisiaeacetyl-CoA synthase. Further genes for acetate kinase and acetyl-CoAsynthase are introduced.

II. Production of Isoprene in Recombinant Strains of Saccharomycescerevisiae

One ml of 15 and 36 hour old cultures of isoprene synthase transformantsdescribed above are transferred to head space vials. The vials aresealed and incubated for 5 hours at 30° C. Head space gas is measuredand isoprene is identified by the methods described above.

Example 14: Construction of Trichoderma reesei Reference and AcetylationModulatory Strains

This Example describes the construction of reference strains and strainscontaining mutations in genes responsible for modulating intracellularprotein acetylation in Trichoderma reesei. This Example also describesmeasuring the effects of an acetyltransferase gene deletion on isopreneproduction in Trichoderma reesei strains expressing the mevalonatepathway and isoprene synthase

I. Construction of Trichoderma reesei Strains Expressing IsopreneSynthase, MVA Pathway and PKL

Genes encoding Trichoderma reesei codon-optimized P. alba or P.tremuloides isoprene synthase, E. gallinarum PKL and E. gallinarum mvaEand mvaS are synthesized by DNA 2.0. Using standard molecular biologytechniques, the genes are cloned into a vector such as pTrex3 g underthe control of sophorose-inducible promoters. pyr2 Trichoderma reeseiprotoplasts are transformed with a mixture of the above constructs andtransformants are selected on amdS selection plates supplemented withuradine. Stable transformants are grown in media containing aglucose-sophorose carbon source and screened for expression of IspS, PKLand the MVA pathway proteins by immunoblot. Strains expressing allproteins are screened for isoprene production. Isoprene producers areselected for spore purification and further manipulation.

II. Construction Strains Containing a Deletion of a Trichoderma reeseiAcetyltransferase Gene

Isoprene-producing strains containing a deletion of a Trichoderma reeseiacetyltransferase are generated using standard molecular biologyprotocols. Briefly, a cassette containing a loxP-flanked the hph gene isinserted within 2-4 kb of chromosomal DNA within or flanking thetargeted acetyltransferase gene. This cassette is transformed into T.reesei protoplasts and hygromycin-resistant transformants are selectedon Vogel's plates. Stable transformants are screened by PCR forinsertion of the cassette to confirm disruption of the targeted locusand subsequently spore-purified. The resulting strain is confirmed toproduce isoprene and be deleted for the acetyltransferase gene.

III. Construction of Strains for Acetate Cycling Via Pta

A gene encoding Trichoderma reesei codon-optimized E. coli pta issynthesized by DNA 2.0. Using standard molecular biology techniques, thegene is cloned under the control of a sophorose-inducible promoter suchas from cbh1 on a hph-marked telomeric vector. This DNA is used totransform protoplasts of isoprene-producing Trichoderma reesei with andwithout the lysine acetyltransferase gene deleted. Transformants areselected on Vogel's plates containing hygromycin.

IV. Construction of Strains for Acetate Cycling Via T. reesei Acs1

A gene encoding Trichoderma reesei codon-optimized E. coli ackA issynthesized by DNA 2.0. Using standard molecular biology techniques, thegene is cloned under the control of a sophorose-inducible promoter suchas from cbh1 on a hph-marked telomeric vector. A second cassetteexpressing the T. reesei acs1 gene from a sophorose-inducible promoteris cloned onto this vector. This DNA is used to transform protoplasts ofisoprene-producing Trichoderma reesei with and without the lysineacetyltransferase gene deleted. Transformants are selected on Vogel'splates containing hygromycin.

V. Production of Isoprene in Recombinant Strains of Trichoderma reesei

Cultures of strains indicated in Table 11 are induced withglucose-sophorose and at 15 and 36 hours one mL from each culture istransferred to head space vials. The vials are sealed and incubated for5 hours at 32° C. Head space gas is measured and isoprene is identifiedby the methods described above. Broth is clarified and MVA is identifiedby HPLC.

TABLE 11 Relevant genotypes of T. reesei strains for acetate recyclewith and without deletion of acetyltransferase E. gallinarum P. alba E.gallinarum T. reesei lysine E. coli E. coli mvaE and mvaS ispS PKLacetyltransferase pta ackA T. reesei acs1 + + + + − − wt + + + − − −wt + + + + + − wt + + + − + − wt + + + + − + overexpressed + + + − − +overexpressed

SEQUENCES L. grayi mvaEatggttaaagacattgtaataattgatgccctccgtactcccatcggtaagtaccgcggtcagctctcaaagatgacggcggtggaattgggaaccgcagttacaaaggctctgttcgagaagaacgaccaggtcaaagaccatgtagaacaagtcatttttggcaacgttttacaggcagggaacggccagaatcccgcccgtcagatcgcccttaattctggcctgtccgcagagataccggcttcgactattaaccaggtgtgtggttctggcctgaaagcaataagcatggcgcgccaacagatcctactcggagaagcggaagtaatagtagcaggaggtatcgaatccatgacgaatgcgccgagtattacatattataataaagaagaagacaccctctcaaagcctgttcctacgatgaccttcgatggtctgaccgacgcgtttagcggaaagattatgggtttaacagccgaaaatgttgccgaacagtacggcgtatcacgtgaggcccaggacgcctttgcgtatggatcgcagatgaaagcagcaaaggcccaagaacagggcattttcgcagctgaaatactgcctcttgaaataggggacgaagttattactcaggacgagggggttcgtcaagagaccaccctcgaaaaattaagtctgcttcggaccatttttaaagaagatggtactgttacagcgggcaacgcctcaacgatcaatgatggcgcctcagccgtgatcattgcatcaaaggagtttgctgagacaaaccagattccctaccttgcgatcgtacatgatattacagagataggcattgatccatcaataatgggcattgctcccgtgagtgcgatcaataaactgatcgatcgtaaccaaattagcatggaagaaatcgatctctttgaaattaatgaggcatttgcagcatcctcggtggtagttcaaaaagagttaagcattcccgatgaaaagatcaatattggcggttccggtattgcactaggccatcctcttggcgccacaggagcgcgcattgtaaccaccctagcgcaccagttgaaacgtacacacggacgctatggtattgcctccctgtgcattggcggtggccttggcctagcaatattaatagaagtgcctcaggaagatcagccggttaaaaaattttatcaattggcccgtgaggaccgtctggctagacttcaggagcaagccgtgatcagcccagctacaaaacatgtactggcagaaatgacacttcctgaagatattgccgacaatctgatcgaaaatcaaatatctgaaatggaaatccctcttggtgtggctttgaatctgagggtcaatgataagagttataccatcccactagcaactgaggaaccgagtgtaatcgctgcctgtaataatggtgcaaaaatggcaaaccacctgggcggttttcagtcagaattaaaagatggtacctgcgtgggcaaattgtacttatgaacgtcaaagaacccgcaactatcgagcatacgatcacggcagagaaagcggcaatttttcgtgccgcagcgcagtcacatccatcgattgtgaaacgaggtgggggtctaaaagagatagtagtgcgtacgttcgatgatgatccgacgttcctgtctattgatctgatagttgatactaaagacgcaatgggcgctaacatcattaacaccattctcgagggtgtagccggattctgagggaaatccttaccgaagaaattctgttctctattttatctaattacgcaaccgaatcaattgtgaccgccagctgtcgcataccttacgaagcactgagtaaaaaaggtgatggtaaacgaatcgctgaaaaagtggctgctgcatctaaatttgcccagttagatccttatcgagctgcaacccacaacaaaggtattatgaatggtattgaggccgtcgttaggcctcaggaaatgacacacgggcggtcgcggcagccgcacatgcgtatgcttcacgcgatcagcactatcggggcttaagccagtggcaggttgcagaaggcgcgttacacggggagatcagtctaccacttgcactcggcagcgttggcggtgcaattgaggtcttgcctaaagcgaaggcggcattcgaaatcatggggatcacagaggcgaaggagctggcagaagtcacagctgcggtagggctggcgcaaaacctggcggcgttaagagcgcttgttagtgaaggaatacagcaaggtcacatgtcgctccaggctcgctctcttgcattatcggtaggtgctacaggcaaggaagttgaaatcctggccgaaaaattacagggctctcgtatgaatcaggcgaacgctcagaccatactcgcagagatcagatcgcaaaaagttgaattgtga SEQ ID NO: 1L. grayi mvaSatgaccatgaacgttggaatcgataaaatgtcattctttgttccaccttactttgtggacatgactgatctggcagtagcacgggatgtcgatcccaataagtttctgattggtattggccaggaccagatggcagttaatccgaaaacgcaggatattgtgacatttgccacaaatgctgccaaaaacatactgtcagctgaggaccttgataaaattgatatggtcatagtcggcaccgagagtggaatcgatgaatccaaagcgagtgccgtagtgcttcacaggttgctcggtatccagaagtttgctcgctcctttgaaatcaaagaagcctgttatgggggtaccgcggctttacagttcgctgtaaaccacattaggaatcatcctgaatcaaaggttcttgtagttgcatcagatatcgcgaaatacggcctggcttctggaggtgaaccaacgcaaggtgcaggcgctgtggctatgctcgtctcaactgaccctaagatcattgctttcaacgacgatagcctcgcgcttacacaagatatctatgacttctggcgaccagttggacatgactatcctatggtcgacgggcctcttagtacagagacctacatccagtcatttcagaccgtatggcaggaatacacaaaacggtcgcagcatgcactggcagactttgctgcccttagctttcatatcccgtatactaaaatgggcaaaaaggcgctgcttgcaatccttgaaggcgaatcagaggaggctcagaaccgtatactagcaaaatatgaaaagagtatagcctactccagaaaggcgggtaacctgtataccggtagcctgtatctaggacttatttcacttctggaaaatgcagaagaccttaaagctggtgatttaataggcctcttttcttacggttccggtgctgttgcggagtttttctcaggaaggctggttgaggactatcaggaacagctacttaaaacaaaacatgccgaacagctggcccatagaaagcaactgacaatcgaggagtacgaaacgatgttctccgatcgcttggacgtggacaaagacgccgaatacgaagacacattagcttatagcatttcgtcagtccgaaacaccgtacgtgagtacaggagttga SEQ ID NO: 2 E. faecium mvaEatgaaagaagtggttatgattgatgcggctcgcacacccattgggaaatacagaggtagtcttagtccttttacagcggtggagctggggacactggtcacgaaagggctgctggataaaacaaagcttaagaaagacaagatagaccaagtgatattcggcaatgtgcttcaggcaggaaacggacaaaacgttgcaagacaaatagccctgaacagtggcttaccagttgacgtgccggcgatgactattaacgaagtttgcgggtccggaatgaaagcggtgattttagcccgccagttaatacagttaggggaggcagagttggtcattgcagggggtacggagtcaatgtcacaagcacccatgctgaaaccttaccagtcagagaccaacgaatacggagagccgatatcatcaatggttaatgacgggctgacggatgcgttttccaatgctcacatgggtcttactgccgaaaaggtggcgacccagttttcagtgtcgcgcgaggaacaagaccggtacgcattgtccagccaattgaaagcagcgcacgcggttgaagccggggtgttctcagaagagattattccggttaagattagcgacgaggatgtcttgagtgaagacgaggcagtaagaggcaacagcactttggaaaaactgggcaccttgcggacggtgttttctgaagagggcacggttaccgctggcaatgcttcaccgctgaatgacggcgctagtgtcgtgattcttgcatcaaaagaatacgcggaaaacaataatctgccttacctggcgacgataaaggaggttgcggaagttggtatcgatccttctatcatgggtattgccccaataaaggccattcaaaagttaacagatcggtcgggcatgaacctgtccacgattgatctgttcgaaattaatgaagcattcgcggcatctagcattgttgtttctcaagagctgcaattggacgaagaaaaagtgaatatctatggcggggcgatagctttaggccatccaatcggcgcaagcggagcccggatactgacaaccttagcatacggcctcctgcgtgagcaaaagcgttatggtattgcgtcattatgtatcggcggtggtcttggtctggccgtgctgttagaagctaatatggagcagacccacaaagacgttcagaagaaaaagttttaccagcttaccccctccgagcggagatcgcagcttatcgagaagaacgttctgactcaagaaacggcacttattttccaggagcagacgttgtccgaagaactgtccgatcacatgattgagaatcaggtctccgaagtggaaattccaatgggaattgcacaaaattttcagattaatggcaagaaaaaatggattcctatggcgactgaagaaccttcagtaatagcggcagcatcgaacggcgccaaaatctgcgggaacatttgcgcggaaacgcctcagcggcttatgcgcgggcagattgtcctgtctggcaaatcagaatatcaagccgtgataaatgccgtgaatcatcgcaaagaagaactgattctttgcgcaaacgagtcgtacccgagtattgttaaacgcgggggaggtgttcaggatatttctacgcgggagtttatgggttcttttcacgcgtatttatcaatcgactttctggtggacgtcaaggacgcaatgggggcaaacatgatcaactctattctcgaaagcgttgcaaataaactgcgtgaatggttcccggaagaggaaatactgttctccatcctgtcaaacttcgctacggagtccctggcatctgcatgttgcgagattccattgaaagacttggtcgtaacaaagaaattggtgaacagatcgccaagaaaattcaacaggcaggggaatatgctaagcttgacccttaccgcgcggcaacccataacaaggggattatgaacggtatcgaagccgtcgttgccgcaacgggaaacgacacacgggctgtttccgcttctattcacgcatacgccgcccgtaatggcttgtaccaaggtttaacggattggcagatcaagggcgataaactggttggtaaattaacagtcccactggctgtggcgactgtcggtggcgcgtcgaacatattaccaaaagccaaagcttccctcgccatgctggatattgattccgcaaaagaactggcccaagtgatcgccgcggtaggtttagcacagaatctggcggcgttacgtgcattagtgacagaaggcattcagaaaggacacatgggcttgcaagcacgttctttagcgatttcgataggtgccatcggtgaggagatagagcaagtcgcgaaaaaactgcgtgaagctgaaaaaatgaatcagcaaacggcaatacagatatagaaaaaattcgcgagaaatga SEQ ID NO: 3E. faecium mvaSatgaaaatcggtattgaccgtctgtccttcttcatcccgaatttgtatttggacatgactgagctggcagaatcacgcggggatgatccagctaaatatcatattggaatcggacaagatcagatggcagtgaatcgcgcaaacgaggacatcataacactgggtgcaaacgctgcgagtaagatcgtgacagagaaagaccgcgagttgattgatatggtaatcgttggcacggaatcaggaattgaccactccaaagcaagcgccgtgattattcaccatctccttaaaattcagtcgttcgcccgttctttcgaggtaaaagaagcttgctatggcggaactgctgccctgcacatggcgaaggagtatgtcaaaaatcatccggagcgtaaggtcttggtaattgcgtcagacatcgcgcgttatggtttggccagcggaggagaagttactcaaggcgtgggggccgtagccatgatgattacacaaaacccccggattattcgattgaagacgatagtgtttttctcacagaggatatctatgatttctggcggcctgattactccgagttccctgtagtggacgggcccctttcaaactcaacgtatatagagagttttcagaaagtttggaaccggcacaaggaattgtccggaagagggctggaagattatcaagctattgcttttcacataccctatacgaagatgggtaagaaagcgctccagagtgttttagaccaaaccgatgaagataaccaggagcgcttaatggctagatatgaggagtctattcgctatagccggagaattggtaacctgtacacaggcagcttgtaccttggtcttacaagcttgttggaaaactctaaaagtttacaaccgggagatcggatcggcctcttttcctatggcagtggtgcggtgtccgagttctttaccgggtatttagaagaaaattaccaagagtacctgttcgctcaaagccatcaagaaatgctggatagccggactcggattacggtcgatgaatacgagaccatcttttcagagactctgccagaacatggtgaatgcgccgaatatacgagcgacgtccccttttctataaccaagattgagaacgacattcgttattataaaatctga SEQ ID NO: 4E. gallinarum mvaEatggaagaagtggtaattatagatgcacgtcggactccgattggtaaatatcacgggtcgttgaagaagttttcagcggtggcgctggggacggccgtggctaaagacatgttcgaacgcaaccagaaaatcaaagaggagatcgcgcaggtcataattggtaatgtcttgcaggcaggaaatggccagaaccccgcgcggcaagttgctcttcaatcagggttgtccgttgacattcccgcttctacaattaacgaggtttgtgggtctggtttgaaagctatcttgatgggcatggaacaaatccaactcggcaaagcgcaagtagtgctggcaggcggcattgaatcaatgacaaatgcgccaagcctgtcccactataacaaggcggaggatacgtatagtgtcccagtgtcgagcatgacactggatggtctgacagacgcattttctagtaaacctatgggattaacagcggaaaacgtcgcacagcgctacggtatctcccgtgaggcgcaagatcaattcgcatatcaatctcagatgaaagcagcaaaagcgcaggcagaaaacaaattcgctaaggaaattgtgccactggcgggtgaaactaaaaccatcacagctgacgaagggatcagatcccaaacaacgatggagaaactggcaagtctcaaacctgtttttaaaaccgatggcactgtaaccgcagggaatgctagcaccattaatgacggggccgcccttgtgctgcttgctagcaaaacttactgcgaaactaatgacataccgtaccttgcgacaatcaaagaaattgttgaagttggaatcgatccggagattatgggcatctctccgataaaagcgatacaaacattgttacaaaatcaaaaagttagcctcgaagatattggagtttttgaaataaatgaagcctttgccgcaagtagcatagtggttgaatctgagttgggattagatccggctaaagttaaccgttatgggggtggtatatccttaggtcatgcaattggggcaaccggcgctcgcctggccacttcactggtgtatcaaatgcaggagatacaagcacgttatggtattgcgagcctgtgcgttggtggtggacttggactggcaatgcttttagaacgtccaactattgagaaggctaaaccgacagacaaaaagttctatgaattgtcaccagctgaacggttgcaagagctggaaaatcaacagaaaatcagttctgaaactaaacagcagttatctcagatgatgcttgccgaggacactgcaaaccatttgatagaaaatcaaatatcagagattgaactcccaatgggcgtcgggatgaacctgaaggttgatgggaaagcctatgttgtgccaatggcgacggaagagccgtccgtcatcgcggccatgtctaatggtgccaaaatggccggcgaaattcacactcagtcgaaagaacggctgctcagaggtcagattgttttcagcgcgaagaatccgaatgaaatcgaacagagaatagctgagaaccaagctttgattttcgaacgtgccgaacagtcctatccttccattgtgaaaagagagggaggtctccgccgcattgcacttcgtcattttcctgccgattctcagcaggagtctgcggaccagtccacattatatcagtggaccatagtagatgtgaaagacgcgatgggggcaaatatcataaatgcaatacttgagggcgtcgcagccctgtttcgcgaatggttccccaatgaggaaattctatactattctctcgaacttggctacggagagcttagtcacggctgtttgtgaagtcccatttagtgcacttagcaagagaggtggtgcaacggtggcccagaaaattgtgcaggcgtcgctcttcgcaaagacagacccataccgcgcagtgacccacaacaaagggattatgaacggtgtagaggctgttatgcttgccacaggcaacgacacgcgcgcagtctcagccgcttgtcatggatacgcagcgcgcaccggtagctatcagggtctgactaactggacgattgagtcggatcgcctggtaggcgagataacactgccgctggccatcgctacagttggaggcgctaccaaagtgttgcccaaagctcaagcggcactggagattagtgatgttcactcttctcaagagcttgcagccttagcggcgtcagtaggtttagtacaaaatctcgcggccctgcgcgcactggtttccgaaggtatacaaaaagggcacatgtccatgcaagcccggtctctcgcaatcgcggtcggtgctgaaaaagccgagatcgagcaggtcgccgaaaagttgcggcagaacccgccaatgaatcagcagcaggcgctccgttacttggcgagatccgcgaacaatga SEQ ID NO: 5E. gallinarum mvaSatgaacgtcggcattgacaaaattaatttttttcgttccaccgtattatctggatatggtcgacctggcccacgcacgcgaagtggacccgaacaaatttacaattggaattggacaggatcagatggctgtgagcaaaaagacgcacgatatcgtaacattcgcggctagtgccgcgaaggaaattttagaacctgaggacttgcaagctatagacatggttatagttggtaccgaatcgggcattgacgagagcaaagcatccgcggtcgattacatcgtttgttgggcgtacaacctttcgctcgcagttttgaaattaaagaagcctgttacggggcaaccgcaggcattcagtttgccaagactcatatacaagcgaacccggagagcaaggtcctggtaattgcaagcgatatagctcggtatggtcttcggtcaggtggagagcccacacaaggcgcaggggcagttgctatgcttctcacggcaaatcccagaatcctgaccttcgaaaacgacaatctgatgttaacgcaggatatttatgacttctggagaccacttggtcacgcttaccctatggtagatggccacctttccaatcaagtctatattgacagttttaagaaggtctggcaagcacattgcgaacgcaatcaagcttctatatccgactatgccgcgattagttttcatattccgtatacaaaaatgggtaagaaagccctgctcgctgtttttgcagatgaagtggaaactgaacaggaacgcgttatggcacggtatgaagagtctatcgtatattcacgccggatcggcaacttgtatacgggatcattgtacctggggctgatatccttattggaaaacagttctcacctgtcggcgggcgaccggataggattgtttagttatgggagtggcgctgtcagcgaatttttctccggtcgtttagtggcaggctatgaaaatcaattgaacaaagaggcgcatacccagctcctggatcagcgtcagaagctttccatcgaagagtatgaggcgatttttacagattccttagaaattgatcaggatgcagcgttctcggatgacctgccatattccatccgcgagataaaaaacacgattcggtactataaggagagctga SEQ ID NO: 6 E. casseliflavus mvaEatggaagaagttgtcatcattgacgcactgcgtactccaataggaaagtaccacggttcgctgaaagattacacagctgttgaactggggacagtagcagcaaaggcgttgctggcacgaaatcagcaagcaaaagaacacatagcgcaagttattattggcaacgtcctgcaagccggaagtgggcagaatccaggccgacaagtcagtttacagtcaggattgtcttctgatatccccgctagcacgatcaatgaagtgtgtggctcgggtatgaaagcgattctgatgggtatggagcaaattcagctgaacaaagcctctgtggtcttaacaggcggaattgaaagcatgaccaacgcgccgctgtttagttattacaacaaggctgaggatcaatattcggcgccggttagcacaatgatgcacgatggtctaacagatgctttcagttccaaaccaatgggcttaaccgcagagaccgtcgctgagagatatggaattacgcgtaaggaacaagatgaatttgcttatcactctcaaatgaaggcggccaaagcccaggcggcgaaaaagtttgatcaggaaattgtacccctgacggaaaaatccggaacggttctccaggacgaaggcatcagagccgcgacaacagtcgagaagctagctgagcttaaaacggtgttcaaaaaagacggaacagttacagcgggtaacgcctctacgataaatgatggcgctgctatggtattaatagcatcaaaatcttattgcgaagaacaccagattccttatctggccgttataaaggagatcgttgaggtgggttttgcccccgaaataatgggtatttcccccattaaggctatagacaccctgctgaaaaatcaagcactgaccatagaggatataggaatatttgagattaatgaagcctttgctgcgagttcgattgtggtagaacgcgagttgggcctggaccccaaaaaagttaatcgctatggcggtggtatatcactcggccacgcaattggggcgacgggagctcgcattgcgacgaccgttgcttatcagctgaaagatacccaggagcgctacggtatagcttccttatgcgttggtgggggtcttggattggcgatgcttctggaaaacccatcggccactgcctcacaaactaattttgatgaggaatctgcttccgaaaaaactgagaagaagaagttttatgcgctagctcctaacgaacgcttagcgtttttggaagcccaaggcgctattaccgctgctgaaaccctggtcttccaggagatgaccttaaacaaagagacagccaatcacttaatcgaaaaccaaatcagcgaagttgaaattcctttaggcgtgggcctgaacttacaggtgaatgggaaagcgtataatgttcctctggccacggaggaaccgtccgttatcgctgcgatgtcgaatggcgccaaaatggctggtcctattacaacaacaagtcaggagaggctgttacggggtcagattgtcttcatggacgtacaggacccagaagcaatattagcgaaagttgaatccgagcaagctaccattttcgcggtggcaaatgaaacatacccgtctatcgtgaaaagaggaggaggtctgcgtagagtcattggcaggaatttcagtccggccgaaagtgacttagccacggcgtatgtatcaattgacctgatggtagatgttaaggatgcaatgggtgctaatatcatcaatagtatcctagaaggtgttgcggaattgtttagaaaatggttcccagaagaagaaatcctgttctcaattctctccaatctcgcgacagaaagtctggtaacggcgacgtgctcagttccgtttgataaattgtccaaaactgggaatggtcgacaagtagctggtaaaatagtgcacgcggcggactttgctaagatagatccatacagagctgccacacacaataaaggtattatgaatggcgttgaagcgttaatcttagccaccggtaatgacacccgtgcggtgtcggctgcatgccacggttacgcggcacgcaatgggcgaatgcaagggcttacctcttggacgattatcgaagatcggctgataggctctatcacattacctttggctattgcgacagtggggggtgccacaaaaatcttgccaaaagcacaggccgccctggcgctaactggcgttgagacggcgtcggaactggccagcctggcggcgagtgtgggattagttcaaaatttggccgctttacgagcactagtgagcgagggcattcagcaagggcacatgagtatgcaagctagatccctggccattagcgtaggtgcgaaaggtactgaaatagagcaactagctgcgaagctgagggcagcgacgcaaatgaatcaggagcaggctcgtaaatttctgaccgaaataagaaattaaSEQ ID NO: 7 E. casseliflavus mvaSatgaacgttggaattgataaaatcaattttttcgttccgccctatttcattgatatggtggatctcgctcatgcaagagaagttgaccccaacaagttcactataggaataggccaagatcagatggcagtaaacaagaaaacgcaagatatcgtaacgttcgcgatgcacgccgcgaaggatattctgactaaggaagatttacaggccatagatatggtaatagtggggactgagtctgggatcgacgagagcaaggcaagtgctgtcgtattgcatcggcttttaggtattcagccttttgcgcgctcctttgaaattaaggaggcatgctatggggccactgccggccttcagtttgcaaaagctcatgtgcaggctaatccccagagcaaggtcctggtggtagcttccgatatagcacgctacggactggcatccggaggagaaccgactcaaggtgtaggtgctgtggcaatgttgatttccgctgatccagctatcttgcagttagaaaatgataatctcatgttgacccaagatatatacgatttttggcgcccggtcgggcatcaatatcctatggtagacggccatctgtctaatgccgtctatatagacagctttaaacaagtctggcaagcacattgcgagaaaaaccaacggactgctaaagattatgctgcattgtcgttccatattccgtacacgaaaatgggtaagaaagctctgttagcggtttttgcggaggaagatgagacagaacaaaagcggttaatggcacgttatgaagaatcaattgtatacagtcgtcggactggaaatctgtatactggctcactctatctgggcctgatttccttactggagaatagtagcagtttacaggcgaacgatcgcataggtctgtttagctatggttcaggggccgttgcggaatttttcagtggcctcttggtaccgggttacgagaaacaattagcgcaagctgcccatcaagctcttctggacgaccggcaaaaactgactatcgcagagtacgaagccatgtttaatgaaaccattgatattgatcaggaccagtcatttgaggatgacttactgtactccatcagagagatcaaaaacactattcgctactataacgaggagaatgaataa SEQ ID NO: 8E. gallinarum EG2 (mvaE)MEEVVIIDARRTPIGKYHGSLKKFSAVALGTAVAKDMFERNQKIKEEIAQVIIGNVLQAGNGQNPARQVALQSGLSVDIPASTINEVCGSGLKAILMGMEQIQLGKAQVVLAGGIESMTNAPSLSHYNKAEDTYSVPVSSMTLDGLTDAFSSKPMGLTAENVAQRYGISREAQDQFAYQSQMKAAKAQAENKFAKEIVPLAGETKTITADEGIRSQTTMEKLASLKPVFKTDGTVTAGNASTINDGAALVLLASKTYCETNDIPYLATIKEIVEVGIDPEIMGISPIKAIQTLLQNQKVSLEDIGVFEINEAFAASSIVVESELGLDPAKVNRYGGGISLGHAIGATGARLATSLVYQMQEIQARYGIASLCVGGGLGLAMLLERPTIEKAKPTDKKFYELSPAERLQELENQQKISSETKQQLSQMMLAEDTANHLIENQISEIELPMGVGMNLKVDGKAYVVPMATEEPSVIAAMSNGAKMAGEIHTQSKERLLRGQIVFSAKNPNEIEQRIAENQALIFERAEQSYPSIVKREGGLRRIALRHFPADSQQESADQSTFLSVDLFVDVKDAMGANIINAILEGVAALFREWFPNEEILFSILSNLATESLVTAVCEVPFSALSKRGGATVAQKIVQASLFAKTDPYRAVTHNKGIMNGVEAVMLATGNDTRAVSAACHGYAARTGSYQGLTNWTIESDRLVGEITLPLAIATVGGATKVLPKAQAALEISDVHSSQELAALAASVGLVQNLAALRALVSEGIQKGHMSMQARSLAIAVGAEKAEIEQVAEKLRQNPPMNQQQALRFLGEIREQ SEQ ID NO: 9E. gallinarum EG2 (mvaS)MNVGIDKINFFVPPYYLDMVDLAHAREVDPNKFTIGIGQDQMAVSKKTHDIVTFAASAAKEILEPEDLQAIDMVIVGTESGIDESKASAVVLHRLLGVQPFARSFEIKEACYGATAGIQFAKTHIQANPESKVLVIASDIARYGLRSGGEPTQGAGAVAMLLTANPRILTFENDNLMLTQDIYDFWRPLGHAYPMVDGHLSNQVYIDSFKKVWQAHCERNQASISDYAAISFHIPYTKMGKKALLAVFADEVETEQERVMARYEESIVYSRRIGNLYTGSLYLGLISLLENSSHLSAGDRIGLFSYGSGAVSEFFSGRLVAGYENQLNKEAHTQLLDQRQKLSIEEYEAIFTDSLEIDQDAAFSDDLPYSIREIKNTIRYYKES SEQ ID NO: 10 L. grayi (mvaE)MVKDIVIIDALRTPIGKYRGQLSKMTAVELGTAVTKALFEKNDQVKDHVEQVIFGNVLQAGNGQNPARQTALNSGLSAEIPASTINQVCGSGLKAISMARQQILLGEAEVIVAGGIESMTNAPSITYYNKEEDTLSKPVPTMTFDGLTDAFSGKIMGLTAENVAEQYGVSREAQDAFAYGSQMKAAKAQEQGIFAAEILPLEIGDEVITQDEGVRQETTLEKLSLLRTIFKEDGTVTAGNASTINDGASAVIIASKEFAETNQIPYLAIVHDITEIGIDPSIMGIAPVSAINKLIDRNQISMEEIDLFEINEAFAASSVVVQKELSIPDEKINIGGSGIALGHPLGATGARIVTTLAHQLKRTHGRYGIASLCIGGGLGLAILIEVPQEDQPVKKFYQLAREDRLARLQEQAVISPATKHVLAEMTLPEDIADNLIENQISEMEIPLGVALNLRVNDKSYTIPLATEEPSVIAACNNGAKMANHLGGFQSELKDGFLRGQIVLMNVKEPATIEHTITAEKAAIFRAAAQSHPSIVKRGGGLKEIVVRTFDDDPTFLSIDLIVDTKDAMGANIINTILEGVAGFLREILTEEILFSILSNYATESIVTASCRIPYEALSKKGDGKRIAEKVAAASKFAQLDPYRAATHNKGIMNGIEAVVLASGNDTRAVAAAAHAYASRDQHYRGLSQWQVAEGALHGEISLPLALGSVGGAIEVLPKAKAAFEIMGITEAKELAEVTAAVGLAQNLAALRALVSEGIQQGHMSLQARSLALSVGATGKEVEILAEKLQGSRMNQANAQTILAEIRSQKVEL SEQ ID NO: 11 L. grayi (mvaS)MTMNVGIDKMSFFVPPYFVDMTDLAVARDVDPNKFLIGIGQDQMAVNPKTQDIVTFATNAAKNILSAEDLDKIDMVIVGTESGIDESKASAVVLHRLLGIQKFARSFEIKEACYGGTAALQFAVNHIRNHPESKYLVVASDIAKYGLASGGEPTQGAGAVAMLVSTDPKITAFNDDSLALTQDIYDFWRPVGHDYPMVDGPLSTETYIQSFQTVWQEYTKRSQHALADFAALSFHIPYTKMGKKALLAILEGESEEAQNRILAKYEKSIAYSRKAGNLYTGSLYLGLISLLENAEDLKAGDLIGLFSYGSGAVAEFFSGRLVEDYQEQLLKTKHAEQLAHRKQLTIEEYETMFSDRLDVDKDAEYEDTLAYSISSVRNTVREYRS SEQ ID NO: 12 E. faecium (mvaE)MKEVVMIDAARTPIGKYRGSLSPFTAVELGTLVTKGLLDKTKLKKDKIDQVIFGNVLQAGNGQNVARQIALNSGLPVDVPAMTINEVCGSGMKAVILARQLIQLGEAELVIAGGTESMSQAPMLKPYQSETNEYGEPISSMVNDGLTDAFSNAHMGLTAEKVATQFSVSREEQDRYALSSQLKAAHAVEAGVFSEEIIPVKISDEDVLSEDEAVRGNSTLEKLGTLRTVFSEEGTVTAGNASPLNDGASVVILASKEYAENNNLPYLATIKEVAEVGIDPSIMGIAPIKAIQKLTDRSGMNLSTIDLFEINEAFAASSIVVSQELQLDEEKVNIYGGAIALGHPIGASGARILTTLAYGLLREQKRYGIASLCIGGGLGLAVLLEANMEQTHKDVQKKKFYQLTPSERRSQLIEKNVLTQETALIFQEQTLSEELSDHMIENQVSEVEIPMGIAQNFQINGKKKWIPMATEEPSVIAAASNGAKICGNICAETPQRLMRGQIVLSGKSEYQAVINAVNHRKEELILCANESYPSIVKRGGGVQDISTREFMGSFHAYLSIDFLVDVKDAMGANMINSILESVANKLREWFPEEEILFSILSNFATESLASACCEIPFERLGRNKEIGEQIAKKIQQAGEYAKLDPYRAATHNKGIMNGIEAVVAATGNDTRAVSASIHAYAARNGLYQGLTDWQIKGDKLVGKLTVPLAVATVGGASNILPKAKASLAMLDIDSAKELAQVIAAVGLAQNLAALRALVTEGIQKGHMGLQARSLAISIGAIGEEIEQVAKKLREAEKMNQQTAIQILEKIREK SEQ ID NO: 13 E. faecium (mvaS)MKIGIDRLSFFIPNLYLDMTELAESRGDDPAKYHIGIGQDQMAVNRANEDIITLGANAASKIVTEKDRELIDMVIVGTESGIDHSKASAVIIHHLLKIQSFARSFEVKEACYGGTAALHMAKEYVKNHPERKVLVIASDIARYGLASGGEVTQGVGAVAMMITQNPRILSIEDDSVFLTEDIYDFWRPDYSEFPVVDGPLSNSTYIESFQKVWNRHKELSGRGLEDYQAIAFHIPYTKMGKKALQSVLDQTDEDNQERLMARYEESIRYSRRIGNLYTGSLYLGLTSLLENSKSLQPGDRIGLFSYGSGAVSEFFTGYLEENYQEYLFAQSHQEMLDSRTRITVDEYETIFSETLPEHGECAEYTSDVPFSITKIENDIRYYKI SEQ ID NO: 14 E. casseliflavus (mvaE)MEEVVIIDALRTPIGKYHGSLKDYTAVELGTVAAKALLARNQQAKEHIAQVIIGNVLQAGSGQNPGRQVSLQSGLSSDIPASTINEVCGSGMKAILMGMEQIQLNKASVVLTGGIESMTNAPLFSYYNKAEDQYSAPVSTMMHDGLTDAFSSKPMGLTAETVAERYGITRKEQDEFAYHSQMKAAKAQAAKKFDQEIVPLTEKSGTVLQDEGIRAATTVEKLAELKTVFKKDGTVTAGNASTINDGAAMVLIASKSYCEEHQIPYLAVIKEIVEVGFAPEIMGISPIKAIDTLLKNQALTIEDIGIFEINEAFAASSIVVERELGLDPKKVNRYGGGISLGHAIGATGARIATTVAYQLKDTQERYGIASLCVGGGLGLAMLLENPSATASQTNFDEESASEKTEKKKFYALAPNERLAFLEAQGAITAAETLVFQEMTLNKETANHLIENQISEVEIPLGVGLNLQVNGKAYNVPLATEEPSVIAAMSNGAKMAGPITTTSQERLLRGQIVFMDVQDPEAILAKVESEQATIFAVANETYPSIVKRGGGLRRVIGRNFSPAESDLATAYVSIDLMVDVKDAMGANIINSILEGVAELFRKWFPEEEILFSILSNLATESLVTATCSVPFDKLSKTGNGRQVAGKIVHAADFAKIDPYRAATHNKGIMNGVEALILATGNDTRAVSAACHGYAARNGRMQGLTSWTHEDRLIGSITLPLAIATVGGATKILPKAQAALALTGVETASELASLAASVGLVQNLAALRALVSEGIQQGHMSMQARSLAISVGAKGTEIEQLAAKLRAATQMNQEQARKFLTEIRN SEQ ID NO: 15E. casseliflavus (mvaS)MNVGIDKINFFVPPYFIDMVDLAHAREVDPNKFTIGIGQDQMAVNKKTQDIVTFAMHAAKDILTKEDLQAIDMVIVGTESGIDESKASAVVLHRLLGIQPFARSFEIKEACYGATAGLQFAKAHVQANPQSKVLVVASDIARYGLASGGEPTQGVGAVAMLISADPAILQLENDNLMLTQDIYDFWRPVGHQYPMVDGHLSNAVYIDSFKQVWQAHCEKNQRTAKDYAALSFHIPYTKMGKKALLAVFAEEDETEQKRLMARYEESIVYSRRTGNLYTGSLYLGLISLLENSSSLQANDRIGLFSYGSGAVAEFFSGLLVPGYEKQLAQAAHQALLDDRQKLTIAEYEAMFNETIDIDQDQSFEDDLLYSIREIKNTIRYYNEENE SEQ ID NO: 16Acetoactyl-CoA-synthaseMTDVRFRIIGTGAYVPERIVSNDEVGAPAGVDDDWITRKTGIRQRRWAADDQATSDLATAAGRAALKAAGITPEQLTVIAVATSTPDRPQPPTAAYVQHHLGATGTAAFDVNAVCSGTVFALSSVAGTLVYRGGYALVIGADLYSRILNPADRKTVVLFGDGAGAMVLGPTSTGTGPIVRRVALHTFGGLTDLIRVPAGGSRQPLDTDGLDAGLQYFAMDGREVRRFVTEHLPQLIKGFLHEAGVDAADISHFVPHQANGVMLDEVFGELHLPRATMHRTVETYGNTGAASIPITMDAAVRAGSFRPGELVLLAGFGGGMAASFALIEW SEQ ID NO: 17 E. faecalis mvaEatgaaaacagtagttattattgatgcattacgaacaccaattggaaaatataaaggcagcttaagtcaagtaagtgccgtagacttaggaacacatgttacaacacaacttttaaaaagacattccactatttctgaagaaattgatcaagtaatctttggaaatgatacaagctggaaatggccaaaatcccgcacgacaaatagcaataaacagcggtttgtctcatgaaattcccgcaatgacggttaatgaggtctgcggatcaggaatgaaggccgttattttggcgaaacaattgattcaattaggagaagcggaagttttaattgctggcgggattgagaatatgtcccaagcacctaaattacaacgttttaattacgaaacagaaagctacgatgcgccatactagtatgatgtatgatggattaacggatgcctttagtggtcaggcaatgggcttaactgctgaaaatgtggccgaaaagtatcatgtaactagagaagagcaagatcaattttctgtacattcacaattaaaagcagctcaagcacaagcagaagggatattcgctgacgaaatagccccattagaagtatcaggaacgcttgtggagaaagatgaagggattcgccctaattcgagcgttgagaagctaggaacgcttaaaacagtttttaaagaagacggtactgtaacagcagggaatgcatcaaccattaatgatggggcttctgctttgattattgcttcacaagaatatgccgaagcacacggtcttccttatttagctattattcgagacagtgtggaagtcggtattgatccagcctatatgggaatttcgccgattaaagccattcaaaaactgttagcgcgcaatcaacttactacggaagaaattgatctgtatgaaatcaacgaagcatttgcagcaacttcaatcgtggtccaaagagaactggctttaccagaggaaaaggtcaacatttatggtggcggtatttcattaggtcatgcgattggtgccacaggtgctcgtttattaacgagtttaagttatcaattaaatcaaaaagaaaagaaatatggagtggcttctttatgtatcggcggtggcttaggactcgctatgctactagagagacctcagcaaaaaaaaaacagccgattttatcaaatgagtcctgaggaacgcctggcttctcttcttaatgaaggccagatttctgctgatacaaaaaaagaatttgaaaatacggctttatcttcgcagattgccaatcatatgattgaaaatcaaatcagtgaaacagaagtgccgatgggcgttggcttacatttaacagtggacgaaactgattatttggtaccaatggcgacagaagagccctcagttattgcggctttgagtaatggtgcaaaaatagcacaaggatttaaaacagtgaatcaacaacgcttaatgcgtggacaaatcgtatttacgatgttgcagatcccgagtcattgattgataaactacaagtaagagaagcggaagattcaacaagcagagttaagttatccatctatcgttaaacggggcggcggcttaagagatttgcaatatcgtacttttgatgaatcatttgtatctgtcgactttttagtagatgttaaggatgcaatgggggcaaatatcgttaacgctatgttggaaggtgtggccgagttgttccgtgaatggtttgcggagcaaaagattttattcagtattttaagtaattatgccacggagtcggttgttacgatgaaaacggctattccagtacacgtttaagtaaggggagcaatggccgggaaattgctgaaaaaattgttttagcttcacgctatgcttcattagatccttatcgggcagtcacgcataacaaaggaatcatgaatggcattgaagctgtagttttagctacaggaaatgatacacgcgctgttagcgcttcttgtcatgcttttgcggtgaaggaaggtcgctaccaaggcttgactagttggacgctggatggcgaacaactaattggtgaaatttcagttccgcttgctttagccacggttggcggtgccacaaaagtcttacctaaatctcaagcagctgctgatttgttagcagtgacggatgcaaaagaactaagtcgagtagtagcggctgttggtttggcacaaaatttagcggcgttacgggccttagtctctgaaggaattcaaaaaggacacatggctctacaagcacgttctttagcgatgacggtcggagctactggtaaagaagttgaggcagtcgctcaacaattaaaacgtcaaaaaacgatgaaccaagaccgagccatggctatataaatgatttaagaaaacaataa SEQ ID NO: 18E. faecalis mvaSAtgacaattgggattgataaaattagtttttttgtgcccccttattatattgatatgacggcactggctgaagccagaaatgtagaccctggaaaatttcatattggtattgggcaagaccaaatggcggtgaacccaatcagccaagatattgtgacatttgcagccaatgccgcagaagcgatcttgaccaaagaagataaagaggccattgatatggtgattgtcgggactgagtccagtatcgatgagtcaaaagcggccgcagttgtcttacatcgtttaatggggattcaacctttcgctcgctctttcgaaatcaaggaagcttgttacggagcaacagcaggcttacagttagctaagaatcacgtagccttacatccagataaaaaagtcttggtcgtagcggcagatattgcaaaatatggcttaaattctggcggtgagcctacacaaggagctggggcggttgcaatgttagttgctagtgaaccgcgcattttggctttaaaagaggataatgtgatgctgacgcaagatatctatgacttttggcgtccaacaggccacccgtatcctatggtcgatggtcctttgtcaaacgaaacctacatccaatcttttgcccaagtctgggatgaacataaaaaacgaaccggtcttgattttgcagattatgatgctttagcgttccatattccttacacaaaaatgggcaaaaaagccttattagcaaaaatctccgaccaaactgaagcagaacaggaacgaattttagcccgttatgaagaaagtatcgtctatagtcgtcgcgtaggaaacttgtatacgggttcactttatctgggactcatttcccttttagaaaatgcaacgactttaaccgcaggcaatcaaattggtttattcagttatggttctggtgctgtcgctgaatttttcactggtgaattagtagctggttatcaaaatcatttacaaaaagaaactcatttagcactgctggataatcggacagaactttctatcgctgaatatgaagccatgtttgcagaaactttagacacagacattgatcaaacgttagaagatgaattaaaatatagtatttctgctattaataataccgttcgttcttatcgaaactaa SEQ ID NO: 19 E. faecalis (mvaE)MKTVVIIDALRTPIGKYKGSLSQVSAVDLGTHVTTQLLKRHSTISEEIDQVIFGNVLQAGNGQNPARQIAINSGLSHEIPAMTVNEVCGSGMKAVILAKQLIQLGEAEVLIAGGIENMSQAPKLQRFNYETESYDAPFSSMMYDGLTDAFSGQAMGLTAENVAEKYHVTREEQDQFSVHSQLKAAQAQAEGIFADEIAPLEVSGTLVEKDEGIRPNSSVEKLGTLKTVFKEDGTVTAGNASTINDGASALIIASQEYAEAHGLPYLAIIRDSVEVGIDPAYMGISPIKAIQKLLARNQLTTEEIDLYEINEAFAATSIVVQRELALPEEKVNIYGGGISLGHAIGATGARLLTSLSYQLNQKEKKYGVASLCIGGGLGLAMLLERPQQKKNSRFYQMSPEERLASLLNEGQISADTKKEFENTALSSQIANHMIENQISETEVPMGVGLHLTVDETDYLVPMATEEPSVIAALSNGAKIAQGFKTVNQQRLMRGQIVFYDVADPESLIDKLQVREAEVFQQAELSYPSIVKRGGGLRDLQYRTFDESFVSVDFLVDVKDAMGANIVNAMLEGVAELFREWFAEQKILFSILSNYATESVVTMKTAIPVSRLSKGSNGREIAEKIVLASRYASLDPYRAVTHNKGIMNGIEAVVLATGNDTRAVSASCHAFAVKEGRYQGLTSWTLDGEQLIGEISVPLALATVGGATKVLPKSQAAADLLAVTDAKELSRVVAAVGLAQNLAALRALVSEGIQKGHMALQARSLAMTVGATGKEVEAVAQQLKRQKTMNQDRAMAILNDLRKQ SEQ ID NO: 20 E. faecalis (mvaS)MTIGIDKISFFVPPYYIDMTALAEARNVDPGKFHIGIGQDQMAVNPISQDIVTFAANAAEAILTKEDKEAIDMVIVGTESSIDESKAAAVVLHRLMGIQPFARSFEIKEACYGATAGLQLAKNHVALHPDKKVLVVAADIAKYGLNSGGEPTQGAGAVAMLVASEPRILALKEDNVMLTQDIYDFWRPTGHPYPMVDGPLSNETYIQSFAQVWDEHKKRTGLDFADYDALAFHIPYTKMGKKALLAKISDQTEAEQERILARYEESIVYSRRVGNLYTGSLYLGLISLLENATTLTAGNQIGLFSYGSGAVAEFFTGELVAGYQNHLQKETHLALLDNRTELSIAEYEAMFAETLDTDIDQTLEDELKYSISAINNTVRSYRN SEQ ID NO: 21MEA P. alba Isoprene synthaseatggaagctcgtcgttctgcgaactacgaacctaacagctgggactatgattacctgctgtcctccgacacggacgagtccatcgaagtatacaaagacaaagcgaaaaagctggaagccgaagttcgtcgcgagattaataacgaaaaagcagaatttctgaccctgctggaactgattgacaacgtccagcgcctgggcctgggttaccgtttcgagtctgatatccgtggtgcgctggatcgcttcgtttcctccggcggcttcgatgcggtaaccaagacttccctgcacggtacggcactgtctttccgtctgctgcgtcaacacggttttgaggtttctcaggaagcgttcagcggcttcaaagaccaaaacggcaacttcctggagaacctgaaggaagatatcaaagctatcctgagcctgtacgaggccagcttcctggctctggaaggcgaaaacatcctggacgaggcgaaggttttcgcaatctctcatctgaaagaactgtctgaagaaaagatcggtaaagagctggcagaacaggtgaaccatgcactggaactgccactgcatcgccgtactcagcgtctggaagcagtatggtctatcgaggcctaccgtaaaaaggaggacgcgaatcaggttctgctggagctggcaattctggattacaacatgatccagtctgtataccagcgtgatctgcgtgaaacgtcccgttggtggcgtcgtgtgggtctggcgaccaaactgcactttgctcgtgaccgcctgattgagagcttctactgggccgtgggtgtagcattcgaaccgcaatactccgactgccgtaactccgtcgcaaaaatgttttctttcgtaaccattatcgacgatatctacgatgtatacggcaccctggacgaactggagctgtttactgatgcagttgagcgttgggacgtaaacgccatcaacgacctgccggattacatgaaactgtgctttctggctctgtataacactattaacgaaatcgcctacgacaacctgaaagataaaggtgagaacatcctgccgtatctgaccaaagcctgggctgacctgtgcaacgctttcctgcaagaagccaagtggctgtacaacaaatctactccgacctttgacgactacttcggcaacgcatggaaatcctcttctggcccgctgcaactggtgttcgcttacttcgctgtcgtgcagaacattaaaaaggaagagatcgaaaacctgcaaaaataccatgacaccatctctcgtccttcccatatcttccgtctgtgcaatgacctggctagcgcgtctgcggaaattgcgcgtggtgaaaccgcaaatagcgtttcttgttacatgcgcactaaaggtatctccgaagaactggctaccgaaagcgtgatgaatctgatcgatgaaacctggaaaaagatgaacaaggaaaaactgggtggtagcctgttcgcgaaaccgttcgtggaaaccgcgatcaacctggcacgtcaatctcactgcacttatcataacggcgacgcgcatacctctccggatgagctgacccgcaaacgcgttctgtctgtaatcactgaaccgattctgccgtttgaacgctaa SEQ ID NO: 22ispAtggactttccgcagcaactcgaagcctgcgttaagcaggccaaccaggcgctgagccgttttatcgccccactgccctttcagaacactcccgtggtcgaaaccatgcagtatggcgcattattaggtggtaagcgcctgcgacctttcctggtttatgccaccggtcatatgtttggcgttagcacaaacacgctggacgcacccgctgctgccgtagagtgtatccacgcttactcattaattcatgatgatttaccggcgatggatgatgacgatctgcgccgcggtttgccgacctgccatgtgaagtttggcgaagcaaacgcgattctcgctggcgacgctttacaaacgctggcgttctcgattctaagcgatgccgatatgccggaagtgtcggatcgcgacagaatttcgatgatttctgaactggcgagcgccagcggtattgccggaatgtgcggtggtcaggcactagatttagacgcggaaggcaaacacgtacctctggacgcgcttgagcgtattcatcgtcataaaaccggcgcattgattcgcgccgccgttcgccttggtgcattaagcgccggagataaagggcgtcgtgctctgccagtactcgacaagtacgcagagagcatcggccttgccttccaggttcaagatgacatcctggatgtggtaggagatactgcaacgttgggaaaacgccagggtgccgaccagcaacttggtaaaagtacctaccctgcacttctgggtcttgagcaagcccggaagaaagcccgggatctgatcgacgatgcccgtcagtcgctgaaacaactggctgaacagtcactcgatacctcggcactggaagcgctagcggactacatcatccagcgtaataaataa SEQ ID NO: 23MEA P. alba isoprene synthaseMEARRSANYEPNSWDYDYLLSSDTDESIEVYKDKAKKLEAEVRREINNEKAEFLTLLELIDNVQRLGLGYRFESDIRGALDRFVSSGGFDAVTKTSLHGTALSFRLLRQHGFEVSQEAFSGFKDQNGNFLENLKEDIKAILSLYEASFLALEGENILDEAKVFAISHLKELSEEKIGKELAEQVNHALELPLHRRTQRLEAVWSIEAYRKKEDANQVLLELAILDYNMIQSVYQRDLRETSRWWRRVGLATKLHFARDRLIESFYWAVGVAFEPQYSDCRNSVAKMFSFVTIIDDIYDVYGTLDELELFTDAVERWDVNAINDLPDYMKLCFLALYNTINEIAYDNLKDKGENILPYLTKAWADLCNAFLQEAKWLYNKSTPTFDDYFGNAWKSSSGPLQLVFAYFAVVQNIKKEEIENLQKYHDTISRPSHIFRLCNDLASASAEIARGETANSVSCYMRTKGISEELATESVMNLIDETWKKMNKEKLGGSLFAKPFVETAINLARQSHCTYHNGDAHTSPDELTRKRVLSVITEPILPFER SEQ ID NO: 24Amorphadiene synthase codon-optimized for E. coliATGAGCCTGACCGAAGAAAAACCGATTCGTCCGATTGCAAATTTTCCGCCTAGCATTTGGGGTGATCAGTTTCTGATTTATGAGAAACAGGTTGAACAGGGCGTTGAGCAGATTGTTAATGATCTGAAAAAAGAAGTTCGCCAGCTGCTGAAAGAAGCACTGGATATTCCGATGAAACATGCCAATCTGCTGAAACTGATTGATGAAATTCAGCGTCTGGGTATCCCGTATCATTTTGAACGTGAAATTGATCATGCCCTGCAGTGCATTTATGAAACCTATGGTGATAATTGGAATGGTGATCGTAGCAGCCTGTGGTTTCGTCTGATGCGTAAACAGGGTTATTATGTTACCTGCGACGTGTTTAACAACTATAAAGATAAAAACGGTGCCTTTAAACAGAGCCTGGCAAATGATGTTGAAGGTCTGCTGGAACTGTATGAAGCAACCAGCATGCGTGTTCCGGGTGAAATTATTCTGGAAGATGCACTGGGTTTTACCCGTAGCCGTCTGAGCATGATGACCAAAGATGCATTTAGCACCAATCCGGCACTGTTTACCGAAATCCAGCGTGCACTGAAACAGCCGCTGTGGAAACGTCTGCCTCGTATTGAAGCAGCACAGTATATTCCGTTTTATCAGCAGCAGGATAGCCATAACAAAACCCTGCTGAAACTGGCAAAACTGGAATTTAATCTGCTGCAGAGCCTGCATAAAGAAGAACTGAGCCACGTTTGTAAATGGTGGAAAGCCTTCGACATCAAAAAAAACGCACCGTGTCTGCGTGATCGTATTGTTGAATGTTATTTTTGGGGTCTGGGTAGCGGTTTTGAACCGCAGTATAGCCGTGCACGTGTGTTTTTTACCAAAGCAGTTGCAGTTATTACCCTGATCGATGATACCTATGACGCATATGGCACCTATGAGGAACTGAAAATCTTTACCGAAGCCGTTGAACGTTGGAGCATTACCTGTCTGGATACCCTGCCGGAATATATGAAACCGATCTATAAACTGTTCATGGACACCTATACCGAGATGGAAGAATTTCTGGCAAAAGAAGGTCGTACCGACCTGTTTAATTGCGGTAAAGAATTTGTGAAAGAATTCGTGCGTAACCTGATGGTTGAAGCAAAATGGGCCAATGAAGGTCATATTCCGACCACCGAAGAACATGATCCGGTTGTGATTATTACCGGTGGTGCAAACCTGCTGACCACCACCTGTTATCTGGGTATGAGCGATATTTTCACCAAAGAAAGCGTTGAATGGGCAGTTAGCGCACCGCCTCTGTTTCGTTATAGCGGTATTCTGGGTCGTCGTCTGAACGATCTGATGACCCATAAAGCAGAACAAGAACGTAAACATAGCAGCAGCAGCCTGGAAAGCTATATGAAAGAATATAACGTGAACGAAGAGTATGCACAGACCCTGATTTACAAAGAAGTTGAGGACGTTTGGAAAGATATCAACCGTGAATATCTGACCACGAAAAACATTCCGCGTCCGCTGCTGATGGCAGTTATTTATCTGTGTCAGTTCCTGGAAGTTCAGTATGCAGGTAAAGATAACTTTACGCGTATGGGCGACGAATATAAACATCTGATTAAAAGCCTGCTGGTGTATCCGATGAGCATTTAA SEQ ID NO: 25 Farnesene synthase codon-optimized for E. coliATGAGCACCCTGCCGATTAGCAGCGTTAGCTTTAGCAGCAGCACCAGTCCGCTGGTTGTTGATGATAAAGTTAGCACCAAACCGGATGTTATTCGTCACACCATGAACTTTAATGCAAGCATTTGGGGTGATCAGTTTCTGACCTATGATGAACCGGAAGATCTGGTGATGAAAAAACAGCTGGTTGAAGAACTGAAAGAAGAAGTTAAAAAAGAGCTGATCACCATCAAAGGTAGCAATGAACCGATGCAGCATGTTAAACTGATTGAACTGATCGATGCCGTTCAGCGTCTGGGTATTGCATATCATTTTGAAGAAGAAATCGAAGAAGCCCTGCAGCATATTCATGTTACCTATGGTGAACAGTGGGTGGATAAAGAAAATCTGCAGAGCATTAGCCTGTGGTTTCGTCTGCTGCGTCAGCAGGGTTTTAATGTTAGCAGCGGTGTGTTTAAAGATTTTATGGACGAGAAAGGCAAATTCAAAGAAAGCCTGTGTAATGATGCACAGGGTATTCTGGCACTGTATGAAGCAGCATTTATGCGTGTTGAAGATGAAACCATTCTGGATAATGCACTGGAATTTACCAAAGTGCACCTGGATATCATTGCAAAAGATCCGAGCTGTGATAGCAGCCTGCGTACCCAGATTCATCAGGCACTGAAACAGCCGCTGCGTCGTCGTCTGGCACGCATTGAAGCACTGCATTATATGCCGATTTATCAGCAAGAAACCAGCCATAATGAAGATCTGCTGAAACTGGCAAAACTGGATTTTAGCGTTCTGCAGTCCATGCACAAAAAAGAACTGAGCCATATTTGTAAATGGTGGAAAGATCTGGATCTGCAGAATAAACTGCCGTATGTTCGTGATCGTGTTGTGGAAGGTTATTTTTGGATTCTGAGCATCTATTATGAACCGCAGCATGCACGTACCCGTATGTTTCTGATGAAAACCTGTATGTGGCTGGTTGTGCTGGATGATACGTTTGATAATTATGGCACCTACGAGGAACTGGAAATCTTTACCCAGGCAGTTGAACGTTGGAGCATTAGTTGTCTGGATATGCTGCCGGAATACATGAAACTGATTTATCAAGAACTGGTGAACCTGCACGTTGAAATGGAAGAAAGTCTGGGCAAAGGTGGTAAAAACATTAGCAATAGTCTGTGTCAGGGTCGTTGGCAGAAAGAACTGGGTAGTCAGATTACCCTGGTTGAAACCAAAATGGCAAAACGTGGTGTTCATGCCCAGCCGCTGGAAGAGTATATGAGCGTTAGCATGGTTACCGGCACCTATGGTCTGATGATTGCACGTAGCTATGTTGGTCGTGGTGATATTGTTACCGAAGATACCTTTAAATGGGTGAGCAGCTATCCGCCTATTATCAAAGCAAGCTGTGTTATTGTTCGCCTGATGGATGATATTGTGAGCCACAAAGAAGAACAAGAACGCGGTCATGTTGCCAGCAGCATTGAATGTTATAGCAAAGAAAGTGGTGCAAGCGAAGAAGAAGCCTGCGAATATATCAGCCGTAAAGTGGAAGATGCCTGGAAAGTTATTAATCGTGAAAGCCTGCGTCCGACCGCAGTTCCGTTTCCGCTGCTGATGCCTGCAATTAACCTGGCACGTATGTGTGAAGTTCTGTATAGCGTTAATGATGGTTTTACCCATGCCGAAGGTGATATGAAATCCTATATGAAAAGCTTCTTCGTGCATCCGATGGTTGTTTAA SEQ ID NO: 26pMCM1225-pCL-Ptrc-Upper_GcMM_163 (Enterococcus gallinarum EG2)cccgtcttactgtcgggaattcgcgttggccgattcattaatgcagattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaacaatttatcagacaatctgtgtgggcactcgaccggaattatcgattaactttattattaaaaattaaagaggtatatattaatgtatcgattaaataaggaggaataaaccatggaagaagtggtaattatagatgcacgtcggactccgattggtaaatatcacgggtcgttgaagaagttttcagcggtggcgctggggacggccgtggctaaagacatgttcgaacgcaaccagaaaatcaaagaggagatcgcgcaggtcataattggtaatgtcttgcaggcaggaaatggccagaaccccgcgcggcaagttgctcttcaatcagggttgtccgttgacattcccgcttctacaattaacgaggtttgtgggtctggtttgaaagctatcttgatgggcatggaacaaatccaactcggcaaagcgcaagtagtgctggcaggcggcattgaatcaatgacaaatgcgccaagcctgtcccactataacaaggcggaggatacgtatagtgtcccagtgtcgagcatgacactggatggtctgacagacgcattttctagtaaacctatgggattaacagcggaaaacgtcgcacagcgctacggtatctcccgtgaggcgcaagatcaattcgcatatcaatctcagatgaaagcagcaaaagcgcaggcagaaaacaaattcgctaaggaaattgtgccactggcgggtgaaactaaaaccatcacagctgacgaagggatcagatcccaaacaacgatggagaaactggcaagtctcaaacctgattaaaaccgatggcactgtaaccgcagggaatgctagcaccattaatgacggggccgcccttgtgctgcttgctagcaaaacttactgcgaaactaatgacataccgtaccttgcgacaatcaaagaaattgttgaagttggaatcgatccggagattatgggcatctctccgataaaagcgatacaaacattgttacaaaatcaaaaagttagcctcgaagatattggagtttttgaaataaatgaagcattgccgcaagtagcatagtggttgaatctgagttgggattagatccggctaaagttaaccgttatgggggtggtatatccttaggtcatgcaattggggcaaccggcgctcgcctggccacttcactggtgtatcaaatgcaggagatacaagcacgttatggtattgcgagcctgtgcgttggtggtggacttggactggcaatgcttttagaacgtccaactattgagaaggctaaaccgacagacaaaaagttctatgaattgtcaccagctgaacggttgcaagagctggaaaatcaacagaaaatcagttctgaaactaaacagcagttatctcagatgatgcttgccgaggacactgcaaaccatttgatagaaaatcaaatatcagagattgaactcccaatgggcgtcgggatgaacctgaaggttgatgggaaagcctatgttgtgccaatggcgacggaagagccgtccgtcatcgcggccatgtctaatggtgccaaaatggccggcgaaattcacactcagtcgaaagaacggctgctcagaggtcagattgttttcagcgcgaagaatccgaatgaaatcgaacagagaatagctgagaaccaagctttgattttcgaacgtgccgaacagtcctatccttccattgtgaaaagagagggaggtctccgccgcattgcacttcgtcattttcctgccgattctcagcaggagtctgcggaccagtccacattatatcagtggaccatagtagatgtgaaagacgcgatgggggcaaatatcataaatgcaatacttgagggcgtcgcagccctgtacgcgaatggttccccaatgaggaaattcttttttctattctctcgaacttggctacggagagcttagtcacggctgtttgtgaagtcccatttagtgcacttagcaagagaggtggtgcaacggtggcccagaaaattgtgcaggcgtcgctcttcgcaaagacagacccataccgcgcagtgacccacaacaaagggattatgaacggtgtagaggctgttatgcttgccacaggcaacgacacgcgcgcagtctcagccgcttgtcatggatacgcagcgcgcaccggtagctatcagggtctgactaactggacgattgagtcggatcgcctggtaggcgagataacactgccgctggccatcgctacagttggaggcgctaccaaagtgttgcccaaagctcaagcggcactggagattagtgatgttcactcttctcaagagcttgcagccttagcggcgtcagtaggtttagtacaaaatctcgcggccctgcgcgcactggtttccgaaggtatacaaaaagggcacatgtccatgcaagcccggtctctcgcaatcgcggtcggtgctgaaaaagccgagatcgagcaggtcgccgaaaagttgcggcagaacccgccaatgaatcagcagcaggcgctccgttttcttggcgagatccgcgaacaatgatctagacgcactaggaggatataccaatgaacgtcggcattgacaaaattaattttttcgttccaccgtattatctggatatggtcgacctggcccacgcacgcgaagtggacccgaacaaatttacaattggaattggacaggatcagatggctgtgagcaaaaagacgcacgatatcgtaacattcgcggctagtgccgcgaaggaaattttagaacctgaggacttgcaagctatagacatggttatagttggtaccgaatcgggcattgacgagagcaaagcatccgcggtcgttttacatcgtttgttgggcgtacaacctacgctcgcagttttgaaattaaagaagcctgttacggggcaaccgcaggcattcagtttgccaagactcatatacaagcgaacccggagagcaaggtcctggtaattgcaagcgatatagctcggtatggtcttcggtcaggtggagagcccacacaaggcgcaggggcagttgctatgcttctcacggcaaatcccagaatcctgaccttcgaaaacgacaatctgatgttaacgcaggatatttatgacttctggagaccacttggtcacgcttaccctatggtagatggccacctttccaatcaagtctatattgacagttttaagaaggtctggcaagcacattgcgaacgcaatcaagcttctatatccgactatgccgcgattagttttcatattccgtatacaaaaatgggtaagaaagccctgctcgctgtttttgcagatgaagtggaaactgaacaggaacgcgttatggcacggtatgaagagtctatcgtatattcacgccggatcggcaacttgtatacgggatcattgtacctggggctgatatccttattggaaaacagttctcacctgtcggcgggcgaccggataggattgtttagttatgggagtggcgctgtcagcgaatttttctccggtcgtttagtggcaggctatgaaaatcaattgaacaaagaggcgcatacccagctcctggatcagcgtcagaagctttccatcgaagagtatgaggcgatttttacagattccttagaaattgatcaggatgcagcgttctcggatgacctgccatattccatccgcgagataaaaaacacgattcggtactataaggagagctgactgcagctggtaccatatgggaattcgaagcttgggcccgaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttaaacggtctccagcttggctgttttggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggccatagcgtttctacaaactctttttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgagcttagtaaagccctcgctagattttaatgcggatgttgcgattacttcgccaactattgcgataacaagaaaaagccagcctttcatgatatatctcccaatttgtgtagggcttattatgcacgcttaaaaataataaaagcagacttgacctgatagtttggctgtgagcaattatgtgcttagtgcatctaacgcttgagttaagccgcgccgcgaagcggcgtcggcttgaacgaattgttagacattatttgccgactaccttggtgatctcgcctacacgtagtggacaaattcttccaactgatctgcgcgcgaggccaagcgatcttcttcttgtccaagataagcctgtctagcttcaagtatgacgggctgatactgggccggcaggcgctccattgcccagtcggcagcgacatccttcggcgcgattttgccggttactgcgctgtaccaaatgcgggacaacgtaagcactacatttcgctcatcgccagcccagtcgggcggcgagttccatagcgttaaggtttcatttagcgcctcaaatagatcctgttcaggaaccggatcaaagagttcctccgccgctggacctaccaaggcaacgctatgttctcttgcttttgtcagcaagatagccagatcaatgtcgatcgtggctggctcgaagatacctgcaagaatgtcattgcgctgccattctccaaattgcagttcgcgcttagctggataacgccacggaatgatgtcgtcgtgcacaacaatggtgacttctacagcgcggagaatctcgctctctccaggggaagccgaagtttccaaaaggtcgttgatcaaagctcgccgcgttgtttcatcaagccttacggtcaccgtaaccagcaaatcaatatcactgtgtggcttcaggccgccatccactgcggagccgtacaaatgtacggccagcaacgtcggttcgagatggcgctcgatgacgccaactacctctgatagttgagtcgatacttcggcgatcaccgcttccctcatgatgtttaactttgttttagggcgactgccctgctgcgtaacatcgttgctgctccataacatcaaacatcgacccacggcgtaacgcgcttgctgcttggatgcccgaggcatagactgtaccccaaaaaaacagtcataacaagccatgaaaaccgccactgcgccgttaccaccgctgcgttcggtcaaggttctggaccagttgcgtgagcgcatacgctacttgcattacagcttacgaaccgaacaggcttatgtccactgggttcgtgccttcatccgtaccacggtgtgcgtcacccggcaaccttgggcagcagcgaagtcgaggcatttctgtcctggctggcgaacgagcgcaaggtttcggtctccacgcatcgtcaggcattggcggccttgctgttcttctacggcaaggtgctgtgcacggatctgccctggcttcaggagatcggaagacctcggccgtcgcggcgcttgccggtggtgctgaccccggatgaagtggttcgcatcctcggttttctggaaggcgagcatcgtttgttcgcccagcttctgtatggaacgggcatgcggatcagtgagggtttgcaactgcgggtcaaggatctggatttcgatcacggcacgatcatcgtgcgggagggcaagggctccaaggatcgggccttgatgttacccgagagcttggcacccagcctgcgcgagcaggggaattaattcccacgggttttgctgcccgcaaacgggctgttctggtgttgctagtttgttatcagaatcgcagatccggcttcagccggtttgccggctgaaagcgctatttcttccagaattgccatgattattccccacgggaggcgtcactggctcccgtgttgtcggcagctttgattcgataagcagcatcgcctgtacaggctgtctatgtgtgactgttgagctgtaacaagttgtctcaggtgttcaatttcatgttctagttgctttgttttactggtttcacctgttctattaggtgttacatgctgttcatctgttacattgtcgatctgttcatggtgaacagctttgaatgcaccaaaaactcgtaaaagctctgatgtatctatctatttacaccgttttcatctgtgcatatggacagttaccctagatatgtaacggtgaacagttgttctacttttgtttgttagtcttgatgcttcactgatagatacaagagccataagaacctcagatccttccgtatttagccagtatgttctctagtgtggttcgttgattgcgtgagccatgagaacgaaccattgagatcatacttactttgcatgtcactcaaaaattttgcctcaaaactggtgagctgaatttttgcagttaaagcatcgtgtagtgattcttagtccgttatgtaggtaggaatctgatgtaatggttgttggtattttgtcaccattcatttttatctggttgttctcaagttcggttacgagatccatttgtctatctagttcaacttggaaaatcaacgtatcagtcgggcggcctcgcttatcaaccaccaatttcatattgctgtaagtgtttaaatctttacttattggtttcaaaacccattggttaagccttttaaactcatggtagttattttcaagcattaacatgaacttaaattcatcaaggctaatctctatatttgccttgtgagttacttttgtgttagttcttttaataaccactcataaatcctcatagagtatttgttttcaaaagacttaacatgttccagattatattttatgaatttttttaactggaaaagataaggcaatatctcttcactaaaaactaattctaatttttcgcttgagaacttggcatagtttgtccactggaaaatctcaaagcctttaaccaaaggattcctgatttccacagttctcgtcatcagctctctggttgctttagctaatacaccataagcattttccctactgatgttcatcatctgagcgtattggttataagtgaacgataccgtccgttctttccttgtagggttttcaatcgtggggttgagtagtgccacacagcataaaattagcttggtttcatgctccgttaagtcatagcgactaatcgctagttcatttgctttgaaaacaactaattcagacatacatctcaattggtctaggtgattttaatcactataccaattgagatgggctagtcaatgataattactagtccttttcctttgagttgtgggtatctgtaaattctgctagacctttgctggaaaacttgtaaattctgctagaccctctgtaaattccgctagacctttgtgtgtattatgtttatattcaagtggttataatttatagaataaagaaagaataaaaaaagataaaaagaatagatcccagccctgtgtataactcactactttagtcagttccgcagtattacaaaaggatgtcgcaaacgctgtttgctcctctacaaaacagaccttaaaaccctaaaggcttaagtagcaccctcgcaagctcgggcaaatcgctgaatattccattgtctccgaccatcaggcacctgagtcgctgtctattcgtgacattcagttcgctgcgctcacggctctggcagtgaatgggggtaaatggcactacaggcgccttttatggattcatgcaaggaaactacccataatacaagaaaagcccgtcacgggcttctcagggcgttttatggcgggtctgctatgtggtgctatctgactttttgctgttcagcagttcctgccctctgattttccagtctgaccacttcggattatcccgtgacaggtcattcagactggctaatgcacccagtaaggcagcggtatcatcaacaggctta SEQ ID NO: 27Amino acid sequence for phosphoketolase from Mycoplasma hominis ATCC 23114MISKIYDDKKYLEKMDKWFRAANYLGVCQMYLRDNPLLKKPLTSNDIKLYPIGHWGTVPGQNFIYTHLNRVIKKYDLNMFYIEGPGHGGQVMISNSYLDGSYSEIYPEISQDEAGLAKMFKRFSFPGGTASHAAPETPGSIHEGGELGYSISHGTGAILDNPDVICAAVVGDGEAETGPLATSWFSNAFINPVNDGAILPILHLNGGKISNPTLLSRKPKEEIKKYFEGLGWNPIFVEWSEDKSNLDMHELMAKSLDKAIESIKEIQAEARKKPAEEATRPTWPMIVLRTPKGWTGPKQWNNEAIEGSFRAHQVPIPVSAFKMEKIADLEKWLKSYKPEELFDENGTIIKEIRDLAPEGLKRMAVNPITNGGIDSKPLKLQDWKKYALKIDYPGEIKAQDMAEMAKFAADIMKDNPSSFRVFGPDETKSNRMFALFNVTNRQWLEPVSKKYDEWISPAGRIIDSQLSEHQCEGFLEGYVLTGRHGFFASYEAFLRVVDSMLTQHMKWIKKASELSWRKTYPSLNIIATSNAFQQDHNGYTHQDPGLLGHLADKRPEIIREYLPADTNSLLAVMNKALTERNVINLIVASKQPREQFFTVEDAEELLEKGYKVVPWASNISENEEPDIVFASSGVEPNIESLAAISLINQEYPHLKIRYVYVLDLLKLRSRKIDPRGISDEEFDKVFTKNKPIIFAFHGFEGLLRDIFFTRSNHNLIAHGYRENGDITTSFDIRQLSEMDRYHIAKDAAEAVYGKDAKAFMNKLDQKLEYHRNYIDEYGYDMPEVVEWKWKNINKEN SEQ ID NO: 28Codon optimized DNA sequence for phosphoketolase from Mycoplasma hominis ATCC 23114atgattagcaaaatctatgatgataaaaagtatctggaaaaaatggataaatggtttcgcgcagcaaattatctgggtgtttgtcagatgtatctgcgtgataatccgctgctgaaaaaaccgctgaccagcaatgatatcaaactgtatccgattggtcattggggcaccgttccgggtcagaattttatctatacccatctgaatcgcgtgatcaagaaatatgatctgaatatgttctacatcgaaggtcctggtcatggtggtcaggttatgattagtaatagctatctggatggcagctatagcgaaatttatccggaaattagccaggatgaagcaggtctggccaaaatgtttaaacgttttagattccgggtggcaccgcaagccatgcagcaccggaaacaccgggtagcattcatgaaggtggtgaactgggttatagcattagccatggcaccggtgcaattctggataacccggatgttatttgtgcagcagttgttggtgatggtgaagcagaaaccggtccgctggcgaccagctggtttagcaatgcctttattaacccggttaatgatggtgccattctgccgattctgcatctgaacggtggtaaaattagcaatccgaccctgctgagccgtaaaccgaaagaagaaatcaaaaaatactttgaaggcctgggctggaatccgatttttgttgaatggtcagaagataagagcaacctggatatgcatgaactgatggcaaaaagcctggataaagccattgaaagcatcaaagaaattcaggcagaagcacgtaaaaaacctgcagaagaagcaacccgtccgacctggccgatgattgttctgcgtaccccgaaaggttggacaggtccgaaacagtggaataatgaagcaattgaaggtagctttcgtgcacatcaggttccgattccggttagcgcctttaaaatggaaaagattgccgatcttgagaaatggctgaaaagctacaaaccggaagaactgtttgatgaaaatggcacgatcataaaagaaatccgtgatctggctccggaaggtctgaaacgtatggcagttaacccgattaccaatggtggtattgatagcaaacctctgaaactgcaggattggaaaaagtacgcactgaaaattgattatccgggtgaaattaaagcacaggatatggccgaaatggccaaatttgcagcagatatcatgaaagataaccctagcagctttcgcgtttttggtccggatgaaaccaaaagcaatcgtatgtttgccctgtttaatgtgaccaatcgtcagtggctggaaccggttagtaagaaatacgatgaatggattagtccggcaggtcgcattattgattcacagctgagcgaacatcagtgtgaaggttttctggaaggttatgttctgaccggtcgtcatggtttttttgcaagctatgaagcatttctgcgtgttgtggatagcatgctgacccaacatatgaaatggatcaaaaaggcaagcgaactgagctggcgtaaaacctatccgagcctgaacattattgcaaccagtaatgcatttcagcaggatcataatggttatacgcatcaggatccgggtctgctgggtcatctggcagataaacgtccagaaattatccgtgaatatctgcctgcagataccaatagcctgctggcggttatgaataaagcactgaccgaacgtaatgtgattaatctgattgttgcaagcaaacagcctcgcgaacagttttttaccgttgaagatgcagaggaactgctggaaaagggttataaagttgttccgtgggcaagcaatattagcgaaaatgaagaaccggatattgtgtttgccagcagcggtgttgaaccgaatatcgaaagtctggcagcaattagcctgatcaatcaagaatatcctcatctgaaaatccgctatgtgtatgtgctggatctgctgaagctgcgtagtcgtaaaatcgatccgcgtggtattagtgatgaagagtttgataaagtgtttaccaaaaacaaaccgattatattgcctttcatggctttgagggactgctgcgcgatattttctttacccgtagcaaccataacctgattgcacatggttatcgtgaaaacggtgatatcacaaccagctttgatattcgtcagctgagtgagatggatcgttatcatattgcaaaagatgctgccgaagccgtgtatggtaaagatgcaaaagcatttatgaacaaactggatcagaaactggaataccaccgcaactatatcgatgagtatggctatgatatgccggaagttgtggaatggaaatggaagaacatcaataaagaaaattaa SEQ ID NO: 29Sequence of pMCS1019gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatcggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggcgcactcccgttctggataatgatttgcgccgacatcataacggttctggcaaatattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaacaatttatcagacaatctgtgtgggcactcgaccggaattatcgattaactttattattaaaaattaaagaggtatatattaatgtatcgattaaataaggaggaataaaccatggaaacgcgtcgttctgcgaactacgaacctaacagctgggactatgattacctgctgtcctccgacacggacgagtccatcgaagtatacaaagacaaagcgaaaaagctggaagccgaagttcgtcgcgagattaataacgaaaaagcagaatttctgaccctgctggaactgattgacaacgtccagcgcctgggcctgggttaccgtttcgagtctgatatccgtggtgcgctggatcgcttcgtttcctccggcggcttcgatgcggtaaccaagacttccctgcacggtacggcactgtctttccgtctgctgcgtcaacacggttttgaggtttctcaggaagcgttcagcggcttcaaagaccaaaacggcaacttcctggagaacctgaaggaagatatcaaagctatcctgagcctgtacgaggccagcttcctggctctggaaggcgaaaacatcctggacgaggcgaaggttttcgcaatctctcatctgaaagaactgtctgaagaaaagatcggtaaagagctggcagaacaggtgaaccatgcactggaactgccactgcatcgccgtactcagcgtctggaagcagtatggtctatcgaggcctaccgtaaaaaggaggacgcgaatcaggttctgctggagctggcaattctggattacaacatgatccagtctgtataccagcgtgatctgcgtgaaacgtcccgttggtggcgtcgtgtgggtctggcgaccaaactgcactttgctcgtgaccgcctgattgagagcttctactgggccgtgggtgtagcattcgaaccgcaatactccgactgccgtaactccgtcgcaaaaatgttttgtttcgtaaccattatcgacgatatctacgatgtatacggcaccctggacgaactggagctgtttactgatgcagttgagcgttgggacgtaaacgccatcaacgacctgccggattacatgaaactgtgctttctggctctgtataacactattaacgaaatcgcctacgacaacctgaaagataaaggtgagaacatcctgccgtatctgaccaaagcctgggctgacctgtgcaacgctacctgcaagaagccaagtggctgtacaacaaatctactccgacctttgacgactacttcggcaacgcatggaaatcctcttctggcccgctgcaactggtgttcgcttacttcgctgtcgtgcagaacattaaaaaggaagagatcgaaaacctgcaaaaataccatgacaccatctctcgtccttcccatatcttccgtctgtgcaatgacctggctagcgcgtctgcggaaattgcgcgtggtgaaaccgcaaatagcgtttcttgttacatgcgcactaaaggtatctccgaagaactggctaccgaaagcgtgatgaatctgatcgatgaatattggaaaaagatgaacaaggaaaaactgggtggtagcctgttcgcgaaaccgttcgtggaaaccgcgatcaacctggcacgtcaatctcactgcacttatcataacggcgacgcgcatacctctccggatgagctgacccgcaaacgcgttctgtctgtaatcactgaaccgattctgccgtttgaacgctaaagatacgcgtaaccccaaggacggtaaaatgattagcaaaatctatgatgataaaaagtatctggaaaaaatggataaatggtttcgcgcagcaaattatctgggtgtttgtcagatgtatctgcgtgataatccgctgctgaaaaaaccgctgaccagcaatgatatcaaactgtatccgattggtcattggggcaccgttccgggtcagaattttatctatacccatctgaatcgcgtgatcaagaaatatgatctgaatatgttctacatcgaaggtcctggtcatggtggtcaggttatgattagtaatagctatctggatggcagctatagcgaaatttatccggaaattagccaggatgaagcaggtctggccaaaatgtttaaacgttttagctttccgggtggcaccgcaagccatgcagcaccggaaacaccgggtagcattcatgaaggtggtgaactgggttatagcattagccatggcaccggtgcaattctggataacccggatgttatttgtgcagcagttgttggtgatggtgaagcagaaaccggtccgctggcgaccagctggtttagcaatgcctttattaacccggttaatgatggtgccattctgccgattctgcatctgaacggtggtaaaattagcaatccgaccctgctgagccgtaaaccgaaagaagaaatcaaaaaatactttgaaggcctgggctggaatccgatttttgttgaatggtcagaagataagagcaacctggatatgcatgaactgatggcaaaaagcctggataaagccattgaaagcatcaaagaaattcaggcagaagcacgtaaaaaacctgcagaagaagcaacccgtccgacctggccgatgattgttctgcgtaccccgaaaggttggacaggtccgaaacagtggaataatgaagcaattgaaggtagctttcgtgcacatcaggttccgattccggttagcgcctttaaaatggaaaagattgccgatcttgagaaatggctgaaaagctacaaaccggaagaactgtttgatgaaaatggcacgatcataaaagaaatccgtgatctggctccggaaggtctgaaacgtatggcagttaacccgattaccaatggtggtattgatagcaaacctctgaaactgcaggattggaaaaagtacgcactgaaaattgattatccgggtgaaattaaagcacaggatatggccgaaatggccaaatttgcagcagatatcatgaaagataaccctagcagctttcgcgtttttggtccggatgaaaccaaaagcaatcgtatgtttgccctgtttaatgtgaccaatcgtcagtggctggaaccggttagtaagaaatacgatgaatggattagtccggcaggtcgcattattgattcacagctgagcgaacatcagtgtgaaggttttctggaaggttatgttctgaccggtcgtcatggtattagcaagctatgaagcatttctgcgtgttgtggatagcatgctgacccaacatatgaaatggatcaaaaaggcaagcgaactgagctggcgtaaaacctatccgagcctgaacattattgcaaccagtaatgcatttcagcaggatcataatggttatacgcatcaggatccgggtctgctgggtcatctggcagataaacgtccagaaattatccgtgaatatctgcctgcagataccaatagcctgctggcggttatgaataaagcactgaccgaacgtaatgtgattaatctgattgttgcaagcaaacagcctcgcgaacagttattaccgttgaagatgcagaggaactgctggaaaagggttataaagttgttccgtgggcaagcaatattagcgaaaatgaagaaccggatattgtgtttgccagcagcggtgttgaaccgaatatcgaaagtctggcagcaattagcctgatcaatcaagaatatcctcatctgaaaatccgctatgtgtatgtgctggatctgctgaagctgcgtagtcgtaaaatcgatccgcgtggtattagtgatgaagagtttgataaagtgtttaccaaaaacaaaccgattatctttgcctttcatggctttgagggactgctgcgcgatattttctttacccgtagcaaccataacctgattgcacatggttatcgtgaaaacggtgatatcacaaccagctttgatattcgtcagctgagtgagatggatcgttatcatattgcaaaagatgctgccgaagccgtgtatggtaaagatgcaaaagcatttatgaacaaactggatcagaaactggaataccaccgcaactatatcgatgagtatggctatgatatgccggaagttgtggaatggaaatggaagaacatcaataaagaaaattaaagtctagttaaagtttaaacggtctccagcttggctgttttggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactctttttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccattagcggcattttgccttcctgattgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttaccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctatagcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttagataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccatttactgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtagtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattatgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggccatttacggttcctggccattgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagcagatcaattcgcgcgcgaaggcgaagcggcatgcatttacgttgacaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtacccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagcgcgaattgatctg SEQ ID NO: 30 DNA sequence of plasmid pMCS826gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatcggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggcgcactcccgttctggataatgatttgcgccgacatcataacggttctggcaaatattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaacaatttatcagacaatctgtgtgggcactcgaccggaattatcgattaactttattattaaaaattaaagaggtatatattaatgtatcgattaaataaggaggaataaaccatggaaacgcgtcgttctgcgaactacgaacctaacagctgggactatgattacctgctgtcctccgacacggacgagtccatcgaagtatacaaagacaaagcgaaaaagctggaagccgaagttcgtcgcgagattaataacgaaaaagcagaatttctgaccctgctggaactgattgacaacgtccagcgcctgggcctgggttaccgtttcgagtctgatatccgtggtgcgctggatcgcttcgtttcctccggcggcttcgatgcggtaaccaagacttccctgcacggtacggcactgtctttccgtctgctgcgtcaacacggttttgaggtttctcaggaagcgttcagcggcttcaaagaccaaaacggcaacttcctggagaacctgaaggaagatatcaaagctatcctgagcctgtacgaggccagcttcctggctctggaaggcgaaaacatcctggacgaggcgaaggttttcgcaatctctcatctgaaagaactgtctgaagaaaagatcggtaaagagctggcagaacaggtgaaccatgcactggaactgccactgcatcgccgtactcagcgtctggaagcagtatggtctatcgaggcctaccgtaaaaaggaggacgcgaatcaggttctgctggagctggcaattctggattacaacatgatccagtctgtataccagcgtgatctgcgtgaaacgtcccgttggtggcgtcgtgtgggtctggcgaccaaactgcactttgctcgtgaccgcctgattgagagcttctactgggccgtgggtgtagcattcgaaccgcaatactccgactgccgtaactccgtcgcaaaaatgttttgtttcgtaaccattatcgacgatatctacgatgtatacggcaccctggacgaactggagctgtttactgatgcagttgagcgttgggacgtaaacgccatcaacgacctgccggattacatgaaactgtgctttctggctctgtataacactattaacgaaatcgcctacgacaacctgaaagataaaggtgagaacatcctgccgtatctgaccaaagcctgggctgacctgtgcaacgctacctgcaagaagccaagtggctgtacaacaaatctactccgacctttgacgactacttcggcaacgcatggaaatcctcttctggcccgctgcaactggtgttcgcttacttcgctgtcgtgcagaacattaaaaaggaagagatcgaaaacctgcaaaaataccatgacaccatctctcgtccttcccatatcttccgtctgtgcaatgacctggctagcgcgtctgcggaaattgcgcgtggtgaaaccgcaaatagcgtttcttgttacatgcgcactaaaggtatctccgaagaactggctaccgaaagcgtgatgaatctgatcgatgaatattggaaaaagatgaacaaggaaaaactgggtggtagcctgttcgcgaaaccgttcgtggaaaccgcgatcaacctggcacgtcaatctcactgcacttatcataacggcgacgcgcatacctctccggatgagctgacccgcaaacgcgttctgtctgtaatcactgaaccgattctgccgtttgaacgctaactgcataaaggaggtaaaaaaacatgattagcaaaatctatgatgataaaaagtatctggaaaaaatggataaatggtacgcgcagcaaattatctgggtgtttgtcagatgtatctgcgtgataatccgctgctgaaaaaaccgctgaccagcaatgatatcaaactgtatccgattggtcattggggcaccgttccgggtcagaattttatctatacccatctgaatcgcgtgatcaagaaatatgatctgaatatgttctacatcgaaggtcctggtcatggtggtcaggttatgattagtaatagctatctggatggcagctatagcgaaatttatccggaaattagccaggatgaagcaggtctggccaaaatgtttaaacgttttagctttccgggtggcaccgcaagccatgcagcaccggaaacaccgggtagcattcatgaaggtggtgaactgggttatagcattagccatggcaccggtgcaattctggataacccggatgttatagtgcagcagttgttggtgatggtgaagcagaaaccggtccgctggcgaccagctggtttagcaatgcctttattaacccggttaatgatggtgccattctgccgattctgcatctgaacggtggtaaaattagcaatccgaccctgctgagccgtaaaccgaaagaagaaatcaaaaaatactttgaaggcctgggctggaatccgatttttgttgaatggtcagaagataagagcaacctggatatgcatgaactgatggcaaaaagcctggataaagccattgaaagcatcaaagaaattcaggcagaagcacgtaaaaaacctgcagaagaagcaacccgtccgacctggccgatgattgttctgcgtaccccgaaaggttggacaggtccgaaacagtggaataatgaagcaattgaaggtagctacgtgcacatcaggttccgattccggttagcgcctttaaaatggaaaagattgccgatcttgagaaatggctgaaaagctacaaaccggaagaactgtttgatgaaaatggcacgatcataaaagaaatccgtgatctggctccggaaggtctgaaacgtatggcagttaacccgattaccaatggtggtattgatagcaaacctctgaaactgcaggattggaaaaagtacgcactgaaaattgattatccgggtgaaattaaagcacaggatatggccgaaatggccaaatttgcagcagatatcatgaaagataaccctagcagctttcgcgtttttggtccggatgaaaccaaaagcaatcgtatgtttgccctgtttaatgtgaccaatcgtcagtggctggaaccggttagtaagaaatacgatgaatggattagtccggcaggtcgcattattgattcacagctgagcgaacatcagtgtgaaggttttctggaaggttatgttctgaccggtcgtcatggtattagcaagctatgaagcatttctgcgtgttgtggatagcatgctgacccaacatatgaaatggatcaaaaaggcaagcgaactgagctggcgtaaaacctatccgagcctgaacattattgcaaccagtaatgcatttcagcaggatcataatggttatacgcatcaggatccgggtctgctgggtcatctggcagataaacgtccagaaattatccgtgaatatctgcctgcagataccaatagcctgctggcggttatgaataaagcactgaccgaacgtaatgtgattaatctgattgttgcaagcaaacagcctcgcgaacagttttttaccgttgaagatgcagaggaactgctggaaaagggttataaagttgttccgtgggcaagcaatattagcgaaaatgaagaaccggatattgtgtttgccagcagcggtgttgaaccgaatatcgaaagtctggcagcaattagcctgatcaatcaagaatatcctcatctgaaaatccgctatgtgtatgtgctggatctgctgaagctgcgtagtcgtaaaatcgatccgcgtggtattagtgatgaagagtttgataaagtgtttaccaaaaacaaaccgattatctttgcctttcatggctttgagggactgctgcgcgatattttctttacccgtagcaaccataacctgattgcacatggttatcgtgaaaacggtgatatcacaaccagctttgatattcgtcagctgagtgagatggatcgttatcatattgcaaaagatgctgccgaagccgtgtatggtaaagatgcaaaagcatttatgaacaaactggatcagaaactggaataccaccgcaactatatcgatgagtatggctatgatatgccggaagttgtggaatggaaatggaagaacatcaataaagaaaattaaagtctagttaaagtttaaacggtctccagcttggctgttttggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtactacaaactcatagtttatttactaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccattagcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctatagcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccatttactgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcataccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttagctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagcagatcaattcgcgcgcgaaggcgaagcggcatgcatttacgttgacaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagcgcgaattgatctg SEQ ID NO: 31 MCS534cgctaactgcataaaggaggtaaaaaaac SEQ ID NO: 32 MCS535gctggagaccgtttaaactttaactagacttta SEQ ID NO: 33 oMCS536taaagtctagttaaagtttaaacggtctccagc SEQ ID NO: 34 MCS537gtttttttacctcctttatgcagttagcg SEQ ID NO: 35 MCS488ttagcgttcaaacggcagaatcgg SEQ ID NO: 36 MCS562ccgtttgaacgctaaAGATACGCGTAACCCCAAGGACGGTAAAatgattagcaaaatctatgatgataaaaagtatctgg SEQ ID NO: 37 MCS504tgtttttttacctcctttgcagtgcgtcctgctgatgtgctcagtatcaccgccagtggtatttacgtcaacaccgccagagataatttatcaccgcagatggttatcttaatacgactcactatagggctc SEQ ID NO: 38 MCS516gactcaagatatacttccatcatgcaaaaaaaaatttgcagtgcatgatgttaatcaaattaaccctcactaaagggcg SEQ IDNO: 39 MCS545gctggtcagaccgacgctggttccggtagggatcagcataataatacgggacatGTTTTTTTACCTCCTTTGCAGTGSEQ ID NO: 40 MCM1033 AGGCTGCCTCGTCATCTCTT SEQ ID NO: 41 MCM1035CAGAATATCGCCACTCTGGG SEQ ID NO: 42 MCM1038ACACGCTATCTGGCAGGAAA SEQ ID NO: 43 MCM1039TTTGACAACATCACAGTGCA SEQ ID NO: 44 MCS580aattaaccctcactaaagggcggc SEQ ID NO: 45 MCS584atattccaccagctatttgttagtgaataaaagtggttgaattatttgctcaggatgtggcattgtcaagggctaatacgactcactatagggctcgaggaag SEQ ID NO: 46 MCM1042tcacagcagaacagttagaaagcgtttaaaatcattcggtcacttctgcgggagaccggtaattaaccctcactaaagggcggc SEQID NO: 47 MCM1043CGCGCCAATTACCGCTATCGATTTTGGTCGCAGTAGTGCTTCCAGTCCTCGCTGACTCATATATTCCACCAGCTATTTGTTAGTG SEQ ID NO: 48 MCM1046gcgggaggaatgcgtggtgcggccttcctacatctaaccgattaaacaacagaggttgctaattaaccctcactaaagggcggc SEQID NO: 49 MCM1048GCGCAGGCGGCGTTTATTTTTACGAAAACGACTTAACCGATGACCCCGACGCGACAGCATATATTCCACCAGCTATTTGTTAGTG SEQ ID NO: 50Salmonella enterica acetyltransferase gi|16503810|emb|CAD05835.1|MSQQGLEALLRPKSIAVIGASMKPHRAGYLMMRNLLAGGFNGPVLPVTPAWKAVLGVMAWPDIASLPFTPDLAILCTNASRNLALLDALGAKGCKTCIILSAPTSQHEELLASARHYKMRLLGPNSLGLLAPWQGLNASFSPVPIKQGKLAFISQSAAVSNTILDWAQQREMGFSYFIALGDSLDIDVDELLDYLARDSKTSAILLYLEQLSDARRFVSAARSASRNKPILVIKSGRSPAAQRLLNTSAGMDPAWDAAIQRAGLLRVQDTHELFSAVETLSHMRPLHGDRLMIISNGAAPAALALDELWSRNGKLATLSEETCLQLRQTLPAHIDIANPLDLCDDASSEHYVKTLDILLASQDFDALMVIHSPSAAAPGTESAHALIETIKRHPRGKFVTLLTNWCGEFSSQEARRLFSEAGLPTYRTPEGTITAFMHMVEYRRNQKQLRETPALPSNLTSNTAEAHNLLQRAIAEGATSLDTHEVQPILHAYGLHTLPTWIASDSAEAVHIAEQIGYPVALKLRSPDIPHKSEVQGVMLYLRTASEVQQAANAIFDRVKMAWPQARIHGLLVQSMANRAGAQELRVVVEHDPVFGPLIMLGEGGVEWRPEEQAVVALPPLNMNLARYLVIQGIKQRKIRARSALRPLDIVGLSQLLVQVSNLIVDCPEIQRLDIHPLLASASEFTALDVTLDIAPFDGDNESRLAVRPYPHQLEEWVEMKNGDRCLFRPILPEDEPQLRQFIAQVTKEDLYYRYFSEINEFTHEDLANMTQIDYDREMAFVAVRRMDNAEEILGVTRAISDPDNVDAEFAVLVRSDLKGLGLGRRLMEKLIAYTRDHGLKRLNGITMPNNRGMVALARKLGFQVDIQLDEGIVGLTLNLAKCDES SEQ ID NO: 51Rhodopseudomonas palustris GCN5 family N-acetyltransferasegi|499473135|ref|WP_011159775.1|MSTYRLSTLLSPGAVAVVGASPRPASLGRAVLTNLREAGFKGQIGVVNPRYPEIGGFKTVGSLAELSFVPDLIVITAPPRSVAKVVAEAGELGVAGAIIISSEMGRGKGSYAEAANRAARKSGIRLIGPNCLGIMIPGVNLNASFAAHMPRRGNLALISQSGAIAAGMVDWAAVKEIGFSGIVSIGDQLDVDIADMLDFYAADLDTRAILLYIEAVTDARKFMSAARAAARVKPVVVVKSGRMAHGAKAAATHTGAFAGADAVYEAAFRRAGMLRVYDLRELFDCAETLGRVSAPRGKRVAILTNGGGIGILAVDRLVELGGEPATLSADLHKKLDAILPTSWSGFNPIDITGDADAERYSATLSMLLADPDNDAILVMNVQTAVASPRDIAREVIRVVGEERVRRTLFKPVFAVWVGAEEAVTHAFDAASIPNYPTEDDAVRSIMNMVRYREAVQLLTEVPPSLPKDFDPDTETARAIVEKALREGRTWLDPLEISGLFAAYQIPMIPTLAATNAEEAVSWASSFLSQGVTVVVKVLSRDIPHKSDIGGVVLNLTSVEAVRVAVNEIMARAAKLRPNARLEGVMVQPMILRPKARELTIGIADDPTFGPVIAFGQGGTGVELIDDRSLALPPLDLPLAESLIARTRVSKLLCAYRDVPEVKRSAVALTLVKLSQMAADLPEIRELDVNPLLADESGVVAIDARVVVRPPERKFAGLGNSHFAVKPYPTEWERHLTVKDGWRVLARPIRPDDEPAIHEFLKHVTPEDLRLRFFAAMKEFSHAFIARLSQIDYARAMAFVAFDEITGEMLGVVRIHSDSIYESGEYAILLRSDLKGKGLGWALMKLIIEYARSEGLHYVCGQVLRENTAMLRMCRDLGFETKTDASEPDILNVRLPLTEEAARAAGSA SEQ ID NO: 52Streptomyces lividans protein acetyl transferase EFD66247MSYASRTLGPMQTSSDRHEYPAHWEADVVLRDGGTARVRPITVDDAERLVSFYEQVSDESKYYRFFAPYPRLSAKDVHRFTHHDFVDRVGLAATIGGEFIATVRYDRIGAGGTPATAPADEAEVAFLVQDAHQGRGVASALLEHIAAVARERGIRRFAAEVLPANNKMIKVFMDAGYTQKRSFEDGVVRLEFDLEPTDRSLAVQYAREHRAEARSVQRLLQPGSVAVVGAGRTPGGVGRSILGNIRDAGYTGRLYAVNRAFPEDMKELDGVPACRSVGDIDGPVDLAVVTVPAEHVPDVVTACGEHGVQGLVVISAGYADSGPEGRERQRALVRHARTYGMRIIGPNAFGIINTSPDVRLNASLAPEMPRAGRIGLFAQSGAIGIALLSRLHRRGGGVTGVTGVSTFVSSGNRADVSGNDVLQYWYDDPQTDVALMYLESIGNPRKFTRLARRTAAAKPLVVVQGARHGGVAPQGHAVRATRLPHATVSALLRQAGVIRVDTITDLVDAGLLLARQPLPAGPRVAILGNSESLGLLTYDACLSEGLRPQPPLDLTTAASADDFHAALARALADDTCDAVVVTAIPTLGEGAAGDAVARGGPALGGGRGPHQARPRGPRGAGRPGGGPVRGGEHGSPDGSGHRGHHRPGG SEQ ID NO: 53Mycobacterium tuberculosis acetyltransferase gi|15608138|ref|NP_215513.1|MDGIAELTGARVEDLAGMDVFQGCPAEGLVSLAASVQPLRAAAGQVLLRQGEPAVSFLLISSGSAEVSHVGDDGVAIIARALPGMIVGEIALLRDSPRSATVTTIEPLTGWTGGRGAFATMVHIPGVGERLLRTARQRLAAFVSPIPVRLADGTQLMLRPVLPGDRERTVHGHIQFSGETLYRRFMSARVPSPALMHYLSEVDYVDHFVWVVTDGSDPVADARFVRDETDPTVAEIAFTVADAYQGRGIGSFLIGALSVAARVDGVERFAARMLSDNVPMRTIMDRYGAVWQREDVGVITTMIDVPGPGELSLGREMVDQINRVARQVIEAVG SEQ ID NO: 54Mycobacterium smegmatis acetyl transferase gi|118468187|ref|YP_889697.1|MAELTEVRAADLAALEFFTGCRPSALEPLATQLRPLKAEPGQVLIRQGDPALTFMLIESGRVQVSHAVADGPPIVLDIEPGLIIGEIALLRDAPRTATVVAAEPVIGWVGDRDAFDTILHLPGMFDRLVRIARQRLAAFITPIPVQVRTGEWFYLRPVLPGDVERTLNGPVEFSSETLYRRFQSVRKPTRALLEYLFEVDYADHFVWVMTEGALGPVIADARFVREGHNATMAEVAFTVGDDYQGRGIGSFLMGALIVSANYVGVQRFNARVLTDNMAMRKIMDRLGAVWVREDLGVVMTEVDVPPVDTVPFEPELIDQIRDATRKVIRAVSQ SEQ ID NO: 55Salmonella enterica NAD-dependent deacetylase gi|16764576|ref|NP_460191.1|MQSRRFHRLSRFRKNKRLLRERLRQRIFFRDRVVPEMMENPRVLVLTGAGISAESGIRTFRAADGLWEEHRVEDVATPEGFARNPGLVQTFYNARRQQLQQPEIQPNAAHLALAKLEEALGDRFLLVTQNIDNLHERAGNRNIIHMHGELLKVRCSQSGQILEWNGDVMPEDKCHCCQFPAPLRPHVVWFGEMPLGMDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHTVELNLEPSQVGSEFEEKHYGPASQVVPEFVDKFLKGL SEQ ID NO: 56Rhodopseudomonas palustris NAD-dependent deacetylasegi|499471434|ref|WP_011158074.1|MIAPSLSSGVEQLGDMIAHASSIVPFTGAGISTESGIPDFRSPGGLWSRNQPIPFDEFVARQDARDEAWRRRFAMEQTFAKARPARGHRALASLYKAGKVPAIITQNIDNLHQVSGFAEHDVVELHGNTTYARCIGCGKRHELDWVREWFFRTGHAPHCTACDEPVKTATVSFGQSMPSDAMRRATELAQHCDLFIAIGSSLVVWPAAGFPMLAKECGAKLVIINREPTEQDEIADLVIRHDIGETLGPFVGN SEQ ID NO: 57Mycobacterium tuberculosis NAD-dependent protein deacylasegi|614103494|sp|P9WGG3.1|NPD_MYCTUMRVAVLSGAGISAESGVPTFRDDKNGLWARFDPYELSSTQGWLRNPERVWGWYLWRHYLVANVEPNDGHRAIAAWQDHAEVSVITQNVDDLHERAGSGAVHHLHGSLFEFRCARCGVPYTDALPEMPEPAIEVEPPVCDCGGLIRPDIVWFGEPLPEEPWRSAVEATGSADVMVVVGTSAIVYPAAGLPDLALARGTAVIEVNPEPTPLSGSATISIRESASQALPGLLERLPALLK SEQ ID NO: 58 acsAUppKD3tcacgacagtaaccgcacctacactgtcatgacattgctcgcccctatgtgtaacaaataaccacactgcccatggtccatatgaatatcctccSEQ ID NO: 59 acsADnGI1.6pKD3Rcaacggtctgcgatgttggcaggaatggtgtgtttgtgaatttggctcatatataattcctcctgctatttgttagtgaataaaagtggttgaattatttgctcaggatgtggcattgtcaagggcgtgtaggctggagctgcttcg SEQ ID NO: 60 CMP534gtgcaaattcacaactcagcgg SEQ ID NO: 61 CMP535caccaacgtatcgggcattgc SEQ ID NO: 62 TS Fortcctaattalgttgacactctatcattg SEQ ID NO: 63 TS Revccatcttgttgagaaataaaagaaaatgcca SEQ ID NO: 64 actP Up Fortttatttctcaacaagatgggcaggctatcgcgatgccatcgtaac SEQ ID NO: 65 actP Up Revggagagattacatgatgcttgtacctcatgcagga SEQ ID NO: 66 actP Down Foraagcatcatgtaatctctccccttccccggtcgcctga SEQ ID NO: 67 actP Down Revagtgtcaacaaaaattaggacgtaaccaccatttactgtctgtgga SEQ ID NO: 68actP Test For ctggcgtagtcgagaagctgcttga SEQ ID NO: 69 actP Test Revgcatagcggaacatgaatttagagt SEQ ID NO: 70 ackA Up Fortttatttctcaacaagatggcggatcgagcatagtcatcatcttgtact SEQ ID NO: 71ackA Up GI Revcggttgatttgtttagtggttgaattatttgctcaggatgtggcatngtcaagggcgaatttgacgactcaatgaatatgtact SEQ IDNO: 72 ackA Down GI Foraccactaaacaaatcaaccgcgtttcccggaggtaacctaaaggaggtaaaaaaacatgtcgagtaagttagtactggttctga SEQID NO: 73 ackA Down Revagtgtcaacaaaaattaggagtacccatgaccagaccttccagc SEQ ID NO: 74ackA Up PL RevatcaccgccagtggtatttangtcaacaccgccagagataatttatcaccgcagatggttatctgaatttgacgactcaatgaatatgtactSEQ ID NO: 75 ackA Down PL Fortaaataccactggcggtgatactgagcacatcagcaggacgcactgcaaaggaggtaaaaaaacatgtcgagtaagttagtactggttctga SEQ ID NO: 76 ackA EX Test For tgcaggcgacggtaacgttcagcat SEQ ID NO: 77ackA EX Test Rev gtggaagatgatcgccggatcgata SEQ ID NO: 78 R6K TS Revagtgtcaacaaaaattaggactgtcagccgttaagtgttcctgtgt SEQ ID NO: 79actP R6K For ggtggttacgcagttcaacctgttgatagtacgta SEQ ID NO: 80actP R6K Rev ggttgaactgcgtaaccaccatttactgtctgtgga SEQ ID NO: 81yfiQ DOWN ForTTTATTTCTCAACAAGATGGGGCCGATTAACATCATCCAGACGAT SEQ ID NO: 82yfiQ DOWN GI1.6 RevCGGTTGATTTGTTTAGTGGTTGAATTATTTGCTCAGGATGTGGCATTGTCAAGGGCTCTTGCCCAACGCGAGGAATCATGAGTA SEQ ID NO: 83 yfiQ DOWN GI1.2 RevCGGTTGATTTGTTTAGTGGTTGAATTATTTGCTCAGGATGTGGCATCGTCAAGGGCTCTTGCCCAACGCGAGGAATCATGAGTA SEQ ID NO: 84 yfiQ UP GI ForACCACTAAACAAATCAACCGCGTTTCCCGGAGGTAACCTAAAGGAGGTAAAAAAACACCGGTCTCCCGCAGAAGTGACCGA SEQ ID NO: 85 yfiQ UP RevACTATCAACAGGTTGAACTGCGCCGTTCGATAGCTGGCTGAACGA SEQ ID NO: 86yfiQ Test For GCATCACGCAGCTCCTGGCGGAACA SEQ ID NO: 87 yfiQ Test RevGCTGAACGTGAATTGAGCAGTCGCT SEQ ID NO: 88 rpe R6K ForTACACACATAAGGAGGTTCCCAATGAAACAGTATCTGATCGCACCTAGCA SEQ ID NO: 89rpe R6K Rev TATTCGAATGTATGCTAGTGGACGTCAATCATTACTCGTGGCTCACTTTCGCCAGTTCA SEQ ID NO: 90 tkt R6K ForCACTAGCATACATTCGAATAAGGAGGAATACTATGTCATCTCGTAAGGAACTGGCGAA SEQ ID NO: 91 tkt R6K RevTATCTCCTTCTTGAGCCGATTATCATTACAGCAGCTCTTTGGCTTTCGCGACA SEQ ID NO: 92rpi R6K For ATCGGCTCAAGAAGGAGATATACATATGACGCAGGACGAACTGAAAAAAGCGGTSEQ ID NO: 93 rpi R6K RevTATTCCTCCTTCAGGACCTTTCATTATTTAACGATCGTTTTGACGCCATC SEQ ID NO: 94tat R6K For AAGGTCCTGAAGGAGGAATAAACCATGACCGATAAACTGACCAGCCTGCGT SEQID NO: 95 tat R6K RevGACCGGTTCATTACAGCAGGTCGCCGATCATTTTCTCCA SEQ ID NO: 96 R6K Plasmid ForCCTGCTGTAATGAACCGGTCTCCCGCAGAAGTGACCGAATGA SEQ ID NO: 97 R6K Plasmid RevGGAACCTCCTTATGTGTGTAAACCTTTAGGTTACCTCCGGGAAACGCGGTTGA SEQ ID NO: 98pfKA tmRNA XAA ForTGAAGCGTCCGTTCAAAGGCGACTGGCTAGACTGCGCGAAAAAACTGTATGCTGCT AACGATGAAAATTATGCTNNNGCTGCATAAAATTAACCCTCACTAAAGGGCG SEQ ID NO: 99pfkA tmRNA Rev GCTTCTGTCATCGGTTTCAGGCTAAAGGAATCTGCCTTTTTCCGAAATCATAATACGACTCACTATAGGGCTC SEQ ID NO: 100 pfkA UP ForTTTATTTCTCAACAAGATGGGTTATCGGCGGTGACGGTTCCTACAT SEQ ID NO: 101pfkA UP Rev AGCATAATTTTCATCGTTAGCAGCATACAGTTTTTTCGCGCAGTCTAGCCAGTCGCCT SEQ ID NO: 102 pfkA DOWN R ForCTAACGATGAAAATTATGCTCGCGCTGCATAATGATTTCGGAAAAAGGCAGATTCCT SEQ ID NO: 103 pfkA DOWN I ForCTAACGATGAAAATTATGCTATTGCTGCATAATGATTTCGGAAAAAGGCAGATTCCT SEQ ID NO: 104pfkA DOWN T For CTAACGATGAAAATTATGCTACGGCTGCATAATGATTTCGGAAAAAGGCAGATTCCT SEQ ID NO: 105 pfkA DOWN RevACTATCAACAGGTTGAACTGCGGTGCGGAGTTATCCGGCAGACGT SEQ ID NO: 106pfkA Test For CTGACATGATCAACCGTGGCGGTA SEQ ID NO: 107 pfkA Test RevGATCGTTCCAGTCATGGATCTGCT SEQ ID NO: 108 ackA overexpression plasmidcggatcgagcatagtcatcatcttgtactgattagacaaaataagacgttgcgcgttggtcatttccattgttgactcctgtatcactctactacggtgaaaaaaaagaaggctgagtatgccttcttttatatgcgtaatcaggggtcaattacaaatcatcaaggaaagttttatccagttgtttgaaggcgcgcttaagcgtgtcagctaatgcctggtaatcaggcttgccttcaacgggtgccaacacctgtccagactcctgcaatttaccgcgaacttcataaaaccagttgaggatagcagggggtaatggcgttacagaacgcttgcccagccaccacaatccctgcatgggtaaacttaaggcgaacagcgcagtggcaactgccggcccaagctgaccgcccagggcaatctgccagcagagagtaaatacggcgatcggcggcataaaacggatcgcataacgcgtcatcttgataacgcgattttcgacaaagaccggggcaaggcgtttttccagcggccacgtctttgagtaatgctgtccccggcgaaacaagctaaaaaaattaacagaacgattatccggcgttgacatgcttcacctcaacttcacatataaagattcaaaaatttgtgcaaattcacaactcagcgggacaacgttcaaaacattttgtcttccatacccactatcaggtatcctttagcagcctgaaggcctaagtagtacatattcattgagtcgtcaaattcgcccttgacttatgccacatcctgagcaaataattcaaccactaaacaaatcaaccgcgtttcccggaggtaacctaaaggaggtaaaaaaacatgtcgagtaagttagtactggttctgaactgcggtagttcttcactgaaatttgccatcatcgatgcagtaaatggtgaagagtacctttctggtttagccgaatgtttccacctgcctgaagcacgtatcaaatggaaaatggacggcaataaacaggaagcggctttaggtgcaggcgccgctcacagcgaagcgctcaactttatcgttaatactattctggcacaaaaaccagaactgtctgcgcagctgactgctatcggtcaccgtatcgtacacggcggcgaaaagtataccagctccgtagtgatcgatgagtctgttattcagggtatcaaagatgcagcttcttttgcaccgctgcacaacccggctcacctgatcggtatcgaagaagctctgaaatctttcccacagctgaaagacaaaaacgttgctgtatttgacaccgcgttccaccagactatgccggaagagtcttacctctacgccctgccttacaacctgtacaaagagcacggcatccgtcgttacggcgcgcacggcaccagccacttctatgtaacccaggaagcggcaaaaatgctgaacaaaccggtagaagaactgaacatcatcacctgccacctgggcaacggtggttccgtttctgctatccgcaacggtaaatgcgttgacacctctatgggcctgaccccgctggaaggtctggtcatgggtaccagttcaacctgttgatagtacgtactaagctctcatgtttcacgtactaagctctcatgtttaacgtactaagctctcatgtttaacgaactaaaccctcatggctaacgtactaagctctcatggctaacgtactaagctctcatgtttcacgtactaagctctcatgtttgaacaataaaattaatataaatcagcaacttaaatagcctctaaggttttaagttttataagaaaaaaaagaatatataaggcttttaaagcttttaaggtttaacggttgtggacaacaagccagggatgtaacgcactgagaagcccttagagcctctcaaagcaattttcagtgacacaggaacacttaacggctgacagtcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtgaaatgaatagttcgacaaagatcgcattggtaattacgttactcgatgccatggggattggccttatcatgccagtcttgccaacgttattacgtgaatttattgcttcggaagatatcgctaaccactttggcgtattgcttgcactttatgcgttaatgcaggttatctttgctccttggcttggaaaaatgtctgaccgatttggtcggcgcccagtgctgttgttgtcattaataggcgcatcgctggattacttattgctggctattcaagtgcgctttggatgctgtatttaggccgtttgctttcagggatcacaggagctactggggctgtcgcggcatcggtcattgccgataccacctcagcttctcaacgcgtgaagtggttcggttggttaggggcaagttttgggcttggtttaatagcggggcctattattggtggttttgcaggagagatttcaccgcatagtcccttttttatcgctgcgttgctaaatattgtcactttccttgtggttatgttttggttccgtgaaaccaaaaatacacgtgataatacagataccgaagtaggggttgagacgcaatcgaattcggtatacatcactttatttaaaacgatgcccattttgttgattatttatttttcagcgcaattgataggccaaattcccgcaacggtgtgggtgctatttaccgaaaatcgttttggatggaatagcatgatggttggcttttcattagcgggtcttggtcttttacactcagtattccaagcctttgtggcaggaagaatagccactaaatggggcgaaaaaacggcagtactgctcgaatttattgcagatagtagtgcatttgccatttagcgtttatatctgaaggttggttagatttccctgttttaattttattggctggtggtgggatcgctttacctgcattacagggagtgatgtctatccaaacaaagagtcatgagcaaggtgctttacagggattattggtgagccttaccaatgcaaccggtgttattggcccattactgtttactgttatttataatcattcactaccaatttgggatggctggatttggattattggtttagcgttttactgtattattatcctgctatcgatgaccttcatgttaacccctcaagctcaggggagtaaacaggagacaagtgcttagttatttcgtcaccaaatgatgttattccgcgaaatataatgaccctcttggatcttaacatttttcccctatcatttaccgtcttcatttgtcattattccagaaaaaatcgcgtcattcgactcatgtctaatccaacacgtgtctctcggcttatcccctgacaccgcccgccgacagcccgcatgggacgattctatcaattcagccgcggagtctagttttatattgcagaatgcgagattgctggtttattataacaatataagttttcattattttcaaaaagggggatttattgtgggtttaggtaagaaattgtctgttgctgtcgccgcttcctttatgagtttaaccatcagtctgccgggtgttcaggccgctgaggatatcaataaccaaaaagcatacaaagaaacgtacggcgtctctcatattacacgccatgatatgctgcagatccctaaacagcagcaaaacgaaaaataccaagtgcctcaattcgatcaatcaacgattaaaaatattgagtctgcaaaaggacttgatgtgtccgacagctggccgctgcaaaacgctgacggaacagtagcagaatacaacggctatcacgttgtgtttgctcttgcgggaagcccgaaagacgctgatgacacatcaatctacatgttttatcaaaaggtcggcgacaactcaatcgacagctggaaaaacgcgggccgtgtctttaaagacagcgataagttcgacgccaacgatccgatcctgaaagatcagacgcaagaatggtccggttctgcaacctttacatctgacggaaaaatccgtttattctacactgactattccggtaaacattacggcaaacaaagcctgacaacagcgcaggtaaatgtgtcaaaatctgatgacacactcaaaatcaacggagtggaagatcacaaaacgatttttgacggagacggaaaaacatatcagaacgttcagcagtttatcgatgaaggcaattatacatccgccgacaaccatacgctgagagaccctcactacgttgaagacaaaggccataaataccttgtattcgaagccaacacgggaacagaaaacggataccaaggcgaagaatctttatttaacaaagcgtactacggcggcggcacgaacttcttccgtaaagaaagccagaagcttcagcagagcgctaaaaaacgcgatgctgagttagcgaacggcgccctcggtatcatagagttaaataatgattacacattgaaaaaagtaatgaagccgctgatcacttcaaacacggtaactgatgaaatcgagcgcgcgaatgttttcaaaatgaacggcaaatggtacttgttcactgattcacgcggttcaaaaatgacgatcgatggtattaactcaaacgatatttacatgcttggttatgtatcaaactctttaaccggcccttacaagccgctgaacaaaacagggcttgtgctgcaaatgggtcttgatccaaacgatgtgacattcacttactctcacttcgcagtgccgcaagccaaaggcaacaatgtggttatcacaagctacatgacaaacagaggcttcttcgaggataaaaaggcaacatttggcccaagcttcttaatcaacatcaaaggcaataaaacatccgttgtcaaaaacagcatcctggagcaaggacagctgacagtcaactaataacagcaaaaagaaaatgccgatacttcattggcattttcattatttctcaacaagatgg SEQ ID NO: 109actP deletion plasmidtaatctctccccttccccggtcgcctgaccggggaatactcttcctctccagcatgcatcaccttttcccaaaatattaaacaaataaactcattaaaaaatgagcgatttttgacagtcgtagaaaatgataatgcagagaatatgccttttctttcttgttaattataaggatattttatgtgctacaatggtttaaataatatgattccctctttgccagattaacgataaccactctgtcacaagtccatcacatacaaagaaaacaaaatcagataattacagaaaacatcataaaagcacgttaattgacaataaagccctctctcttttcaagatggatgatcatgaaaaagtgataggcttgattcagaaaatgaaaagaatttatgatagtttaccatcaggaaaaatcacgaaggaaacggacaggaaaatacataaacattttatagatatagctttatatgcaaataataaatgtgacgatagaattacgagaagagtttaccttagtaaagaaaaggaagtatccattaaggtggtatattattataaataatgtcgccatccataataatactatcgaaattccacagacagtaaatggtggttacgcagttcaacctgttgatagtacgtactaagctctcatgtttcacgtactaagctctcatgtttaacgtactaagctctcatgtttaacgaactaaaccctcatggctaacgtactaagctctcatggctaacgtactaagctctcatgtttcacgtactaagctctcatgtttgaacaataaaattaatataaatcagcaacttaaatagcctctaaggttttaagttttataagaaaaaaaagaatatataaggcttttaaagcttttaaggtttaacggttgtggacaacaagccagggatgtaacgcactgagaagcccttagagcctctcaaagcaattttcagtgacacaggaacacttaacggctgacagtcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtgaaatgaatagttcgacaaagatcgcattggtaattacgttactcgatgccatggggattggccttatcatgccagtcttgccaacgttattacgtgaatttattgcttcggaagatatcgctaaccactttggcgtattgcttgcactttatgcgttaatgcaggttatctttgctccttggcttggaaaaatgtctgaccgatttggtcggcgcccagtgctgttgttgtcattaataggcgcatcgctggattacttattgctggctttttcaagtgcgctttggatgctgtatttaggccgtttgctttcagggatcacaggagctactggggctgtcgcggcatcggtcattgccgataccacctcagcttctcaacgcgtgaagtggttcggttggttaggggcaagttagggcttggtttaatagcggggcctattattggtggttttgcaggagagatttcaccgcatagtcccttttttatcgctgcgttgctaaatattgtcactttccttgtggttatgttttggttccgtgaaaccaaaaatacacgtgataatacagataccgaagtaggggttgagacgcaatcgaattcggtatacatcactttatttaaaacgatgcccattttgttgattatttatttttcagcgcaattgataggccaaattcccgcaacggtgtgggtgctatttaccgaaaatcgttttggatggaatagcatgatggttggcttttcattagcgggtcttggtcttttacactcagtattccaagcctttgtggcaggaagaatagccactaaatggggcgaaaaaacggcagtactgctcgaatttattgcagatagtagtgcatttgcctttttagcgtttatatctgaaggttggttagatttccctgttttaattttattggctggtggtgggatcgctttacctgcattacagggagtgatgtctatccaaacaaagagtcatgagcaaggtgctttacagggattattggtgagccttaccaatgcaaccggtgttattggcccattactgtttactgttatttataatcattcactaccaatttgggatggctggatttggattattggtttagcgttttactgtattattatcctgctatcgatgaccttcatgttaacccctcaagctcaggggagtaaacaggagacaagtgcttagttatttcgtcaccaaatgatgttattccgcgaaatataatgaccctcttggatcttaacatttttcccctatcatttttccgtcttcatttgtcattttttccagaaaaaatcgcgtcattcgactcatgtctaatccaacacgtgtctctcggcttatcccctgacaccgcccgccgacagcccgcatgggacgattctatcaattcagccgcggagtctagttttatattgcagaatgcgagattgctggtttattataacaatataagttttcattattttcaaaaagggggatttattgtgggtttaggtaagaaattgtctgttgctgtcgccgcttcctttatgagtttaaccatcagtctgccgggtgttcaggccgctgaggatatcaataaccaaaaagcatacaaagaaacgtacggcgtctctcatattacacgccatgatatgctgcagatccctaaacagcagcaaaacgaaaaataccaagtgcctcaattcgatcaatcaacgattaaaaatattgagtctgcaaaaggacttgatgtgtccgacagctggccgctgcaaaacgctgacggaacagtagcagaatacaacggctatcacgttgtgtttgctcttgcgggaagcccgaaagacgctgatgacacatcaatctacatgttttatcaaaaggtcggcgacaactcaatcgacagctggaaaaacgcgggccgtgtctttaaagacagcgataagttcgacgccaacgatccgatcctgaaagatcagacgcaagaatggtccggttctgcaacctttacatctgacggaaaaatccgtttattctacactgactattccggtaaacattacggcaaacaaagcctgacaacagcgcaggtaaatgtgtcaaaatctgatgacacactcaaaatcaacggagtggaagatcacaaaacgatttttgacggagacggaaaaacatatcagaacgttcagcagtttatcgatgaaggcaattatacatccgccgacaaccatacgctgagagaccctcactacgttgaagacaaaggccataaataccttgtattcgaagccaacacgggaacagaaaacggataccaaggcgaagaatctttatttaacaaagcgtactacggcggcggcacgaacttcttccgtaaagaaagccagaagcttcagcagagcgctaaaaaacgcgatgctgagttagcgaacggcgccctcggtatcatagagttaaataatgattacacattgaaaaaagtaatgaagccgctgatcacttcaaacacggtaactgatgaaatcgagcgcgcgaatgttttcaaaatgaacggcaaatggtacttgttcactgattcacgcggttcaaaaatgacgatcgatggtattaactcaaacgatatttacatgcttggttatgtatcaaactctttaaccggcccttacaagccgctgaacaaaacagggcttgtgctgcaaatgggtcttgatccaaacgatgtgacattcacttactctcacttcgcagtgccgcaagccaaaggcaacaatgtggttatcacaagctacatgacaaacagaggcttcttcgaggataaaaaggcaacatttggcccaagcttcttaatcaacatcaaaggcaataaaacatccgttgtcaaaaacagcatcctggagcaaggacagctgacagtcaactaataacagcaaaaagaaaatgccgatacttcattggcattttcttttatttctcaacaagatgggcaggctatcgcgatgccatcgtaacccacaattgccggatgcgagtcggtaacggtttgtaggcctgataagacgcgacagcgtcgcatcaggcattgattgccggatgcggcgtataacgccttatccggcctacattcggcaagggttacccgagcgttaaccttctcccataagggagcgggaattaaaacaatccctacattacctctggagaatctgtgatgaatggtacgatttatcagcggatagaagacaatgcgcatttcagggagttagtcgaaaaacggcaacggtttgccaccatcctgtcgattattatgctggcagtttatatcggctttattttactgatcgccttcgcgcccggctggctgggcaccccgctgaatccgaacaccagcgtcacacgcggtattccgattggtgttggagtgattgtgatctcctttgttctcaccggtatctacatctggcgggcgaacggcgaattcgaccgtcttaataacgaagtcctgcatgaggtacaagcatcatg SEQ ID NO: 110Pentose Phosphate Pathway Upregulation Plasmidggccgattaacatcatccagacgattaacgccgcggccattcataatattctgtgtaacccattcaaacataatgtctgacatcttacggttacggataagatgataacggtcgtagcgatatttatcgtgctgatgcaggtaaacatcgttcaggctggcaccgctataaagtacgctatcgtcgatgataaagcctttaaagtgcagaacaccaagggcttcacgggtattgattggaacgccataaaccggaacatctacgcccggattttcctgcgccatgcggcagtaccagtcagcgttagtgttagatgccgcagcgccaatgcgtccacgttgtgcacgatgccagtcgaccagcacccgcacatccagttccggacgctgccttttagcttcatacaacgcgttcagaatgcctttgccaccgtcatcctgttcgagatacagggcgacaatgcaaatgcgctgcttcgcgctggctattttttccagcagcgtctcccggaagtcggcgggagcgtaaaagaaatcgacatcatcaactgattgagaaatcttgggtagttgggcaaggtgttgttgatgtttattacgcttaaattttgacaacatcacagtgcatttcttctctgttcattgaagggtcctctgtgcaatgcagacgacataagcgggcaataataacaccagtcccgattaagtggtcaacatttccagtaccttactcatgattcctcgcgttgggcaagagcccttgacttatgccacatcctgagcaaataattcaaccactaaacaaatcaaccgcgtttcccggaggtaacctaaaggtttacacacataaggaggttcccaatgaaacagtatctgatcgcacctagcatcctgtctgcagacttcgcccgtctgggcgaggacaccgctaaagcactggcggcgggcgcggatgtagttcatttcgacgtgatggataatcactacgtaccgaacctgactatcggcccgatggtactgaaatctctgcgtaactacggcatcaccgcgccgatcgatgttcacctgatggttaaaccggtggatcgtatcgtgccggatttcgccgcggctggtgcatccattatcaccttccaccctgaagcttccgaacacgttgaccgcaccctgcagctgattaaagaaaacggctgtaaggctggtctggtgttcaacccagctacgccgctgtcttatctggattatgttatggataagctggacgtaatcctgctgatgagcgtcaacccgggtttcggtggtcagagcttcattccgcagaccctggacaaactgcgcgaagtgcgtcgtcgtatcgatgaatctggcttcgatatccgtctggaggtggatggcggcgtgaaagttaacaacatcggcgagatcgccgcggcaggtgcggacatgttcgtcgcaggttctgcaatcttcgatcagccggactataaaaaagtgattgacgaaatgcgttctgaactggcgaaagtgagccacgagtaatgattgacgtccactagcatacattcgaataaggaggaatactatgtcatctcgtaaggaactggcgaatgccatccgtgctctgtctatggacgccgtgcagaaagccaaatctggtcaccctggcgcaccgatgggcatggcagacatcgccgaagtactgtggcgcgacttcctgaaacataacccgcagaacccgtcttgggctgaccgtgatcgtttcgtgctgagcaacggccacggtagcatgctgatttattccctgctgcacctgactggttacgacctgccgatggaagagctgaagaactttcgccagctgcactccaaaaccccgggtcacccagaagtgggctataccgccggtgtcgagactaccaccggtccactgggccagggcatcgccaacgctgtgggtatggcgattgcagagaagaccctggcagcccagttcaaccgtccgggtcacgatatcgttgaccactatacctacgcctttatgggtgacggctgcatgatggaaggtattagccacgaagtttgctctctggctggcactctgaaactgggtaaactgatcgcattctacgacgacaatggcattagcatcgacggccacgtggaaggttggttcaccgacgacactgcgatgcgtttcgaagcttacggttggcacgtgattcgtgacattgatggtcacgacgcagcgtctatcaaacgtgcggttgaagaggcacgtgccgtaaccgataagccttctctgctgatgtgcaaaacgattatcggtttcggcagcccgaacaaagccggcacccacgacagccatggcgcgcctctgggcgatgccgaaatcgctctgacccgtgagcaactgggttggaaatacgcgccgttcgaaatcccatctgaaatttatgctcagtgggacgcaaaggaagcgggtcaagcaaaggaatctgcatggaacgaaaaatttgctgcttatgctaaggcgtacccgcaggaagcagctgaatttacccgtcgtatgaaaggtgaaatgccgtctgattttgacgcgaaagcgaaggaatttattgcgaaactgcaggcaaacccggcaaaaatcgcctcccgtaaagcgtcccagaacgcgatcgaggcattcggcccgctgctgccggaattcctgggtggttctgccgacctggcgcctagcaacctgaccctgtggtctggttccaaagcaattaatgaagatgctgccggtaactacatccactacggcgtccgcgaatttggtatgaccgcaatcgctaacggtatcagcctgcatggcggttttctgccgtacaccagcacctttctgatgttcgtagaatacgcacgtaacgcggttcgcatggccgcactgatgaaacagcgccaggtgatggtatatactcacgacagcatcggtctgggtgaagacggtccgacccaccagccggttgaacaagttgcgagcctgcgcgtaactccaaacatgtccacgtggcgtccgtgcgaccaggttgaaagcgctgtcgcttggaaatatggcgtggaacgccaggacggtccgaccgcactgatcctgtcccgtcagaatctggctcagcaggagcgtaccgaggagcagctggcaaacatcgcacgtggcggttacgttctgaaagattgcgctggccagccggaactgattttcatcgcaaccggctctgaagtcgagctggcagtcgcagcgtatgagaaactgaccgcggaaggtgttaaagcgcgtgttgtcagcatgccgagcaccgacgcattcgacaaacaggatgcagcatatcgcgagagcgttctgcctaaagctgttactgctcgtgtcgcggttgaggctggtatcgcggactactggtataaatatgtaggtctgaacggtgcgattgttggtatgacgaccttcggtgaatccgctcctgcggaactgctgttcgaagaattcggcttcaccgtagacaacgttgtcgcgaaagccaaagagctgctgtaatgataatcggctcaagaaggagatatacatatgacgcaggacgaactgaaaaaagcggttggttgggcagccctgcagtatgtgcaaccgggtactattgttggtgttggcaccggctccaccgccgcccactttattgatgcgctgggcaccatgaagggtcagatcgaaggtgctgtgtctagctctgacgcgtctactgaaaaactgaagtccctgggcatccacgtgttcgatctgaacgaagttgactctctgggcatctatgtggacggcgcagacgaaattaacggtcacatgcagatgatcaaaggcggtggcgcggccctgacccgcgagaaaatcatcgcatccgttgcagaaaaattcatctgtatcgctgacgcgtctaaacaggtagacattctgggtaaattccctctgccagttgaagtgatccctatggcccgctccgccgtggcccgtcagctggtaaagctgggtggtcgtcctgaatatcgccagggcgttgttactgataacggcaatgtgatcctggacgtgcacggtatggaaatcctggacccgattgcaatggaaaacgcgatcaacgcgattccgggcgttgtaacggtgggcctgttcgcgaatcgcggtgcggacgttgcactgatcggtaccccggatggcgtcaaaacgatcgttaaataatgaaaggtcctgaaggaggaataaaccatgaccgataaactgaccagcctgcgtcagtacaccaccgtagttgcggataccggtgacatcgctgcgatgaaactgtatcaaccgcaggatgcaaccactaacccgtccctgattctgaacgcggcacagatcccggaatatcgtaaactgatcgatgacgcagttgcatgggcaaaacaacagagcaatgatcgcgcccaacagattgtagacgctaccgataaactggccgtaaacatcggcctggagattctgaaactggttccgggtcgtatcagcactgaagttgatgctcgtctgagctatgacacggaagcgagcattgccaaagctaaacgtctgatcaaactgtacaacgacgcgggtatcagcaacgaccgtattctgattaaactggcttctacctggcagggcattcgcgcggccgaacagctggagaaagaaggcatcaactgcaacctgaccctgctgttctcttttgctcaggcccgtgcctgcgctgaagccggtgtttttctgatctctcctttcgtgggccgtattctggattggtacaaagccaacacggataaaaaggagtacgctccggctgaagatccgggtgtggtgagcgtttccgaaatttaccagtactacaaagaacatggttacgaaaccgttgttatgggtgcctcttttcgtaacatcggtgaaatcctggaactggcaggctgcgaccgcctgaccatcgcgccgaccctgctgaaagaactggcggagtctgaaggtgccatcgaacgtaaactgtcctacaccggtgaagtgaaagcacgcccggcacgcattaccgaatctgagttcctgtggcaacacaaccaggatccgatggcagtcgataaactggctgaaggtatccgcaaattcgcaatcgaccaggagaaactggagaaaatgatcggcgacctgctgtaatgaaccggtctcccgcagaagtgaccgaatgattttaaacgctttctaactgttctgctgtgatgctacccagatgttgcgtttttcctgccagatagcgtgttttaaagcgggtaaaatgctcgcctaaccctgctgccgccccggtatcgccggccatatctaacagtgcgatggctacctcggcagtacaatattggccttcagcctgggcttcacgcaggcgataggcagaaagccgggaaagatcgacggaaatgacgggaagattatccagatacggacttttacgaaacatcttgcgagcttccggccaggtaccatcgagcatgataaacagcggtggcttaccggcaggtggtgtgaagatcacttcccgttgctcatcagcatacgaggcgggaaagaccaccattggctgataatacgggttttgtaccagatccagcaaatcctgcgagggttcggtacgcgaccattgaaacgcaacggtatcaggcaaaatatcagcaatcagacgcccggtattactgggcttcattggctcggtgtcgaacatcagcaaacagaagcgactttttgcttgtgctggggtaattgtcgaacagagacataatttctctggcaaaagacagcgttggcagcgacgaacgcgattaccgcgggcaagaaaaggacgtgttgcgcgcgcaatacgctcggcgcgtaactggagaacagcgttttcggtcataagagagcgtcgaaaaaacgccattgtcgcagaggagaaaacggggcacaagatgcgccccggtaagattaaagagattcgttcagccagctatcgaacggcgcagttcaacctgttgatagtacgtactaagctctcatgtttcacgtactaagctctcatgtttaacgtactaagctctcatgtttaacgaactaaaccctcatggctaacgtactaagctctcatggctaacgtactaagctctcatgtttcacgtactaagctctcatgtttgaacaataaaattaatataaatcagcaacttaaatagcctctaaggttttaagttttataagaaaaaaaagaatatataaggcttttaaagcttttaaggtttaacggttgtggacaacaagccagggatgtaacgcactgagaagcccttagagcctctcaaagcaattttcagtgacacaggaacacttaacggctgacagtcctaatttttgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtgaaatgaatagttcgacaaagatcgcattggtaattacgttactcgatgccatggggattggccttatcatgccagtcttgccaacgttattacgtgaatttattgcttcggaagatatcgctaaccactttggcgtattgcttgcactttatgcgttaatgcaggttatctttgctccttggcttggaaaaatgtctgaccgatttggtcggcgcccagtgctgttgttgtcattaataggcgcatcgctggattacttattgctggctttttcaagtgcgctttggatgctgtatttaggccgtttgctttcagggatcacaggagctactggggctgtcgcggcatcggtcattgccgataccacctcagcttctcaacgcgtgaagtggttcggttggttaggggcaagttttgggcttggtttaatagcggggcctattattggtggttttgcaggagagatttcaccgcatagtcccttttttatcgctgcgttgctaaatattgtcactttccttgtggttatgttttggttccgtgaaaccaaaaatacacgtgataatacagataccgaagtaggggttgagacgcaatcgaattcggtatacatcactttatttaaaacgatgcccattttgttgattatttatttttcagcgcaattgataggccaaattcccgcaacggtgtgggtgctatttaccgaaaatcgttttggatggaatagcatgatggttggcttttcattagcgggtcttggtcttttacactcagtattccaagcctttgtggcaggaagaatagccactaaatggggcgaaaaaacggcagtactgctcgaatttattgcagatagtagtgcatttgccatttagcgtttatatctgaaggttggttagatttccctgttttaattttattggctggtggtgggatcgctttacctgcattacagggagtgatgtctatccaaacaaagagtcatgagcaaggtgctttacagggattattggtgagccttaccaatgcaaccggtgttattggcccattactgtttactgttatttataatcattcactaccaatttgggatggctggatttggattattggtttagcgttttactgtattattatcctgctatcgatgaccttcatgttaacccctcaagctcaggggagtaaacaggagacaagtgcttagttatttcgtcaccaaatgatgttattccgcgaaatataatgaccctcttggatcttaacatttttcccctatcatttttccgtcttcatttgtcattttttccagaaaaaatcgcgtcattcgactcatgtctaatccaacacgtgtctctcggcttatcccctgacaccgcccgccgacagcccgcatgggacgattctatcaattcagccgcggagtctagttttatattgcagaatgcgagattgctggtttattataacaatataagttttcattattttcaaaaagggggatttattgtgggtttaggtaagaaattgtctgttgctgtcgccgcttcctttatgagtttaaccatcagtctgccgggtgttcaggccgctgaggatatcaataaccaaaaagcatacaaagaaacgtacggcgtctctcatattacacgccatgatatgctgcagatccctaaacagcagcaaaacgaaaaataccaagtgcctcaattcgatcaatcaacgattaaaaatattgagtctgcaaaaggacttgatgtgtccgacagctggccgctgcaaaacgctgacggaacagtagcagaatacaacggctatcacgttgtgtttgctcttgcgggaagcccgaaagacgctgatgacacatcaatctacatgttttatcaaaaggtcggcgacaactcaatcgacagctggaaaaacgcgggccgtgtctttaaagacagcgataagttcgacgccaacgatccgatcctgaaagatcagacgcaagaatggtccggttctgcaacctttacatctgacggaaaaatccgtttattctacactgactattccggtaaacattacggcaaacaaagcctgacaacagcgcaggtaaatgtgtcaaaatctgatgacacactcaaaatcaacggagtggaagatcacaaaacgatttttgacggagacggaaaaacatatcagaacgttcagcagtttatcgatgaaggcaattatacatccgccgacaaccatacgctgagagaccctcactacgttgaagacaaaggccataaataccttgtattcgaagccaacacgggaacagaaaacggataccaaggcgaagaatctttatttaacaaagcgtactacggcggcggcacgaacttcttccgtaaagaaagccagaagcttcagcagagcgctaaaaaacgcgatgctgagttagcgaacggcgccctcggtatcatagagttaaataatgattacacattgaaaaaagtaatgaagccgctgatcacttcaaacacggtaactgatgaaatcgagcgcgcgaatgttttcaaaatgaacggcaaatggtacttgttcactgattcacgcggttcaaaaatgacgatcgatggtattaactcaaacgatatttacatgcttggttatgtatcaaactctttaaccggcccttacaagccgctgaacaaaacagggcttgtgctgcaaatgggtcttgatccaaacgatgtgacattcacttactctcacttcgcagtgccgcaagccaaaggcaacaatgtggttatcacaagctacatgacaaacagaggcttcttcgaggataaaaaggcaacatttggcccaagcttcttaatcaacatcaaaggcaataaaacatccgttgtcaaaaacagcatcctggagcaaggacagctgacagtcaactaataacagcaaaaagaaaatgccgatacttcattggcattttcttttatttctcaacaagatgg SEQ ID NO: 111pfkA tmRNA allelic exchange vectorgttatcggcggtgacggttcctacatgggtgcaatgcgtctgaccgaaatgggcttcccgtgcatcggcctgccgggcactatcgacaacgacatcaaaggcactgactacactatcggtttcttcactgcgctgagcaccgttgtagaagcgatcgaccgtctgcgtgacacctcttcttctcaccagcgtatttccgtggtggaagtgatgggccgttattgtggcgatctgacgttggctgcggccattgccggtggctgtgaattcgttgtggttccggaagttgaattcagccgtgaagacctggtaaacgaaatcaaagcgggtatcgcgaaaggtaaaaaacacgcgatcgtggcgattaccgaacatatgtgtgatgttgacgaactggcgcatttcatcgagaaagaaaccggtcgtgaaacccgcgcaactgtgctgggccacatccagcgcggtggttctccggtgccttacgaccgtattctggcttcccgtatgggcgcttacgctatcgatctgctgctggcaggttacggcggtcgttgcgtaggtatccagaacgaacagctggttcaccacgacatcatcgacgctatcgaaaacatgaagcgtccgttcaaaggcgactggctagactgcgcgaaaaaactgtatgctgctaacgatgaaaattatgctnnngctgcataatgatttcggaaaaaggcagattcctttagcctgaaaccgatgacagaagcaaaaatgcctgatgcgcttcgcttatcaggcctacgtgaattctgcaatttattgaatttacaaattatgtaggtcggataaggcgttcgcgccgcatccggcatcgataaagcgcactttgtcagcaatatgaggcggatttcttccgccatttaattcctcaacatatacccgcaagttatagccaatctttttttattctttaatgtttggttaaccttctggcacgattgctcatcacaacacaacataagagagtcgggcgatgaacaagtggggcgtagggttaacatttttgctggcggcaaccagcgttatggcaaaggatattcagcttcttaacgtttcatatgatccaacgcgcgaattgtacgaacagtacaacaaggcattcagcgcccactggaaacagcaaactggcgataacgtggtgatccgtcagtcccacggtggttcaggcaaacaagcgacgtcggtaatcaacggtattgaagctgatgttgtcacgctggctctggcctatgacgtggacgcaattgcggaacgcgggcggattgataaagagtggatcaaacgtctgccggataactccgcaccgcagttcaacctgttgatagtacgtactaagctctcatgtttcacgtactaagctctcatgtttaacgtactaagctctcatgtttaacgaactaaaccctcatggctaacgtactaagctctcatggctaacgtactaagctctcatgtttcacgtactaagctctcatgtttgaacaataaaattaatataaatcagcaacttaaatagcctctaaggttttaagttttataagaaaaaaaagaatatataaggcttttaaagcttttaaggtttaacggttgtggacaacaagccagggatgtaacgcactgagaagcccttagagcctctcaaagcaattttcagtgacacaggaacacttaacggctgacagtcctaattatgttgacactctatcattgatagagttattttaccactccctatcagtgatagagaaaagtgaaatgaatagttcgacaaagatcgcattggtaattacgttactcgatgccatggggattggccttatcatgccagtcttgccaacgttattacgtgaatttattgcttcggaagatatcgctaaccactttggcgtattgcttgcactttatgcgttaatgcaggttatctttgctccttggcttggaaaaatgtctgaccgatttggtcggcgcccagtgctgttgttgtcattaataggcgcatcgctggattacttattgctggctttttcaagtgcgctttggatgctgtatttaggccgtttgctttcagggatcacaggagctactggggctgtcgcggcatcggtcattgccgataccacctcagcttctcaacgcgtgaagtggttcggttggttaggggcaagttttgggcttggtttaatagcggggcctattattggtggttttgcaggagagatttcaccgcatagtcccttttttatcgctgcgttgctaaatattgtcactttccttgtggttatgttttggttccgtgaaaccaaaaatacacgtgataatacagataccgaagtaggggttgagacgcaatcgaattcggtatacatcactttatttaaaacgatgcccattttgttgattatttatttttcagcgcaattgataggccaaattcccgcaacggtgtgggtgctatttaccgaaaatcgttttggatggaatagcatgatggttggcttttcattagcgggtcttggtcttttacactcagtattccaagcctttgtggcaggaagaatagccactaaatggggcgaaaaaacggcagtactgctcgaatttattgcagatagtagtgcatttgcctttttagcgtttatatctgaaggttggttagatttccctgttttaattttattggctggtggtgggatcgctttacctgcattacagggagtgatgtctatccaaacaaagagtcatgagcaaggtgctttacagggattattggtgagccttaccaatgcaaccggtgttattggcccattactgtttactgttatttataatcattcactaccaatttgggatggctggatttggattattggtttagcgttttactgtattattatcctgctatcgatgaccttcatgttaacccctcaagctcaggggagtaaacaggagacaagtgcttagttatttcgtcaccaaatgatgttattccgcgaaatataatgaccctcttggatcttaacatttttcccctatcatttttccgtcttcatttgtcattttttccagaaaaaatcgcgtcattcgactcatgtctaatccaacacgtgtctctcggcttatcccctgacaccgcccgccgacagcccgcatgggacgattctatcaattcagccgcggagtctagttttatattgcagaatgcgagattgctggtttattataacaatataagttttcattattttaaaaagggggatttattgtgggtttaggtaagaaattgtctgttgctgtcgccgcttcctttatgagtttaaccatcagtctgccgggtgttcaggccgctgaggatatcaataaccaaaaagcatacaaagaaacgtacggcgtctctcatattacacgccatgatatgctgcagatccctaaacagcagcaaaacgaaaaataccaagtgcctcaattcgatcaatcaacgattaaaaatattgagtctgcaaaaggacttgatgtgtccgacagctggccgctgcaaaacgctgacggaacagtagcagaatacaacggctatcacgttgtgtttgctcttgcgggaagcccgaaagacgctgatgacacatcaatctacatgttttatcaaaaggtcggcgacaactcaatcgacagctggaaaaacgcgggccgtgtctttaaagacagcgataagttcgacgccaacgatccgatcctgaaagatcagacgcaagaatggtccggttctgcaacctttacatctgacggaaaaatccgtttattctacactgactattccggtaaacattacggcaaacaaagcctgacaacagcgcaggtaaatgtgtcaaaatctgatgacacactcaaaatcaacggagtggaagatcacaaaacgatttttgacggagacggaaaaacatatcagaacgttcagcagtttatcgatgaaggcaattatacatccgccgacaaccatacgctgagagaccctcactacgttgaagacaaaggccataaataccttgtattcgaagccaacacgggaacagaaaacggataccaaggcgaagaatattatttaacaaagcgtactacggcggcggcacgaacttcttccgtaaagaaagccagaagcttcagcagagcgctaaaaaacgcgatgctgagttagcgaacggcgccctcggtatcatagagttaaataatgattacacattgaaaaaagtaatgaagccgctgatcacttcaaacacggtaactgatgaaatcgagcgcgcgaatgttttcaaaatgaacggcaaatggtacttgttcactgattcacgcggttcaaaaatgacgatcgatggtattaactcaaacgatatttacatgcttggttatgtatcaaactctttaaccggcccttacaagccgctgaacaaaacagggcttgtgctgcaaatgggtcttgatccaaacgatgtgacattcacttactctcacttcgcagtgccgcaagccaaaggcaacaatgtggttatcacaagctacatgacaaacagaggcttcttcgaggataaaaaggcaacatttggcccaagcttcttaatcaacatcaaaggcaataaaacatccgttgtcaaaaacagcatcctggagcaaggacagctgacagtcaactaataacagcaaaaagaaaatgccgatacttcattggcattacttttatttctcaacaagatgg SEQ IDNO: 112 PL.6 constitutive promoter-lambda promoter, GenBank NC_001416aattcatataaaaaacatacagataaccatctgcggtgataaattatctctggcggtgttgacataaataccactggcggtgatactgagcacatcagcaggacgcactgaccaccatgaaggtg SEQ ID NO: 113

What is claimed is:
 1. Recombinant cells capable of producing anisoprenoid precursor, wherein the cells: (i) (a) have been modified suchthat the activity of a YfiQ polypeptide is modulated by decreasing,attenuating, or deleting the expression of a nucleic acid encoding theYfiQ polypeptide as compared to cells that have not been modified forsuch modulation, and (b) comprise a heterologous nucleic acid encoding aCobB polypeptide, wherein the cells have been modified such that theactivity of the CobB polypeptide is modulated by increasing theexpression of the nucleic acid encoding the CobB polypeptide as comparedto cells that have not been modified for such modulation; and (ii)comprise two or more heterologous nucleic acids encoding two or morepolypeptides of a mevalonate (MVA) pathway, wherein culturing of therecombinant cells in a suitable media provides for the production of theisoprenoid precursor; and (iii) comprise a heterologous nucleic acidencoding a polyprenyl pyrophosphate synthase polypeptide, whereinculturing of the recombinant cells in a suitable media provides forproduction of the isoprenoid.
 2. The recombinant cells of claim 1,wherein the recombinant cells further comprise: (iv) one or more nucleicacids encoding one or more acetyltransferases selected from the groupconsisting of a protein acetyltransferase (Pat) polypeptide and anacetoin utilization protein AcuA (AcuA) polypeptide, wherein theactivity of the one or more acetyltransferases is modulated bydecreasing, attenuating, or deleting the expression of the one or morenucleic acids encoding the one or more acetyltransferases as compared tocells that have not been modified for such modulation; and/or (v) anucleic acid encoding a sirtuin NAD-dependent deacetylase (SrtN)polypeptide, wherein the activity of the SrtN polypeptide is modulatedby increasing the expression of the nucleic acid encoding the SrtNpolypeptide as compared to cells that have not been modified for suchmodulation.
 3. The recombinant cells of claim 1, wherein the two or morepolypeptides of the MVA pathway is selected from (a) an enzyme thatcondenses two molecules of acetyl-CoA to form acetoacetyl-CoA; (b) anenzyme that condenses acetoacetyl-CoA with acetyl-CoA to form3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA); (c) an enzyme thatconverts HMG-CoA to mevalonate; (d) an enzyme that phosphorylatesmevalonate to mevalonate 5-phosphate; (e) an enzyme that convertsmevalonate 5-phosphate to mevalonate 5-pyrophosphate; and (f) an enzymethat converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate.4. The recombinant cells of claim 1, wherein the recombinant cellsfurther comprise (iii) one or more heterologous nucleic acids encoding apolypeptide having phosphoketolase activity.
 5. The recombinant cells ofclaim 1, wherein the recombinant cells further comprise (iii) one ormore nucleic acids encoding one or more pentose phosphate pathwayproteins, wherein the cells have been modified such that the expressionof the one or more nucleic acids encoding the one or more pentosephosphate pathway proteins and/or the activity of the one or morepentose phosphate pathway proteins is modulated relative to recombinantcells that have not been modified.
 6. The recombinant cells of claim 5,wherein the one or more pentose phosphate pathway proteins is selectedfrom the group consisting of transketolase (tktA), transaldolase (talB),ribulose-5-phosphate-epimerase (rpe), ribose-5-phosphate epimerase(rpiA), and phosphofructokinase (pfkA).
 7. The recombinant cells ofclaim 1, wherein the recombinant cells further comprise (iii) one ormore nucleic acids encoding one or more acetate cycling proteins,wherein the cells have been modified such that the expression of the oneor more nucleic acids encoding the one or more acetate cycling proteinsand/or activity of the one or more acetate cycling proteins is modulatedrelative to recombinant cells that have not been modified.
 8. Therecombinant cells of claim 7, wherein the one or more acetate cyclingproteins is selected from the group consisting of acetyl-coenzyme Asynthetase (acs), acetate kinase (ackA), acetate transporter/acetatepump (actP) and phosphotransacetylase (pta).
 9. The recombinant cells ofclaim 1, wherein the recombinant cells further comprise one or morenucleic acids encoding one or more proteins selected from the groupconsisting of malic enzyme (sfcA), malic enzyme (maeB), pyruvatedehydrogenase complex repressor (pdhR), pyruvate decarboxylase (aceE),pyruvate decarboxylase (aceF), pyruvate decarboxylase (lpdA), citratesynthase (glta), acetyl-coenzyme A synthetase (acs), phosphateacetyltransferase (pta), acetate kinase (ackA), acetatetransporter/acetate pump (actP), phosphofructokinase (pfkA),ribulose-5-phosphate-epimerase (rpe), ribose-5-phosphate epimerase(rpiA), transketolase (tkta), transaldolase (talB),6-phosphogluconolactonase (pgl), phosphogluconate dehydratase (edd), and2-keto-3-deoxygluconate 6-phosphate aldolase (eda), and wherein thecells have been modified such that the expression of the one or morenucleic acids and/or activity of the one or more proteins is modulatedrelative to recombinant cells that have not been modified.
 10. Therecombinant cells of claim 1, wherein the recombinant cells aregram-positive bacterial cells, gram-negative bacterial cells, fungalcells, filamentous fungal cells, algal cells, or yeast cells.
 11. Therecombinant cells of claim 10, wherein the recombinant cells areselected from the group consisting of Bacillus subtilis, Streptomyceslividans, Streptomyces coelicolor, Streptomyces griseus, Escherichiacoli, and Pantoea citrea.
 12. The recombinant cells of claim 10, whereinthe recombinant cells are selected from the group consisting ofTrichoderma reesei, Aspergillus oryzae, Aspergillus niger, Saccharomycescerevisiae, and Yarrowia lipolytica.
 13. The recombinant cells of claim1, wherein the isoprenoid precursor is selected from the groupconsisting of mevalonate (MVA), dimethylallyl diphosphate (DMAPP), andisopentenyl pyrophosphate (IPP).
 14. The recombinant cells of 1, whereinthe isoprenoid precursor production is increased relative to recombinantcells that have not been modified in (i)(a) and (i)(b).
 15. Therecombinant cells of claim 14, wherein the isoprenoid precursorproduction is increased by at least 5%, wherein the increased productionof the isoprenoid precursor comprises an increase in: (i) titer, (ii)instantaneous yield, (iii) cumulative yield, (iv) specific productivity,or (v) cell productivity index.
 16. A method of producing an isoprenoidprecursor comprising: (a) culturing the recombinant cell of claim 1under conditions suitable for producing the isoprenoid precursor and (b)producing the isoprenoid precursor.
 17. The method of claim 16, furthercomprising (c) recovering the isoprenoid precursor.
 18. Recombinantcells capable of producing an isoprenoid, wherein the cells: (i) (a)have been modified such that the activity of a YfiQ polypeptide ismodulated by decreasing, attenuating, or deleting the expression of anucleic acid encoding the YfiQ polypeptide as compared to cells thathave not been modified for such modulation, and (b) comprise aheterologous nucleic acid encoding a CobB polypeptide, wherein the cellshave been modified such that the activity of the CobB polypeptide ismodulated by increasing the expression of the nucleic acid encoding theCobB polypeptide as compared to cells that have not been modified forsuch modulation; (ii) comprise two or more heterologous nucleic acidsencoding two or more polypeptides of the MVA pathway; and (iii) comprisea heterologous nucleic acid encoding a polyprenyl pyrophosphate synthasepolypeptide, wherein culturing of the recombinant cells in a suitablemedia provides for production of the isoprenoid.
 19. The recombinantcells of claim 18, wherein the recombinant cells further comprise: (iv)one or more nucleic acids encoding one or more acetyltransferasesselected from the group consisting of a protein acetyltransferase (Pat)polypeptide and an acetoin utilization protein AcuA (AcuA) polypeptide,wherein the activity of the one or more acetyltransferases is modulatedby decreasing, attenuating, or deleting the expression of the one ormore nucleic acids encoding the one or more acetyltransferases ascompared to cells that have not been modified for such modulation;and/or (v) a nucleic acid encoding a sirtuin NAD-dependent deacetylase(SrtN) polypeptide, wherein the activity of the SrtN polypeptide ismodulated by increasing the expression of the nucleic acid encoding theSrtN polypeptide as compared to cells that have not been modified forsuch modulation.
 20. The recombinant cells of claim 1, wherein the twoor more polypeptides of the MVA pathway is selected from (a) an enzymethat condenses two molecules of acetyl-CoA to form acetoacetyl-CoA; (b)an enzyme that condenses acetoacetyl-CoA with acetyl-CoA to form3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA); (c) an enzyme thatconverts HMG-CoA to mevalonate; (d) an enzyme that phosphorylatesmevalonate to mevalonate 5-phosphate; (e) an enzyme that convertsmevalonate 5-phosphate to mevalonate 5-pyrophosphate; and (f) an enzymethat converts mevalonate 5-pyrophosphate to isopentenyl pyrophosphate.21. The recombinant cells of claim 18, wherein the recombinant cellsfurther comprise (iv) one or more heterologous nucleic acids encoding apolypeptide having phosphoketolase activity.
 22. The recombinant cellsof claim 18, wherein the recombinant cells further comprise (iv) one ormore nucleic acids encoding one or more pentose phosphate pathwayproteins, wherein the cells have been modified such that the expressionof the one or more nucleic acids encoding the one or more pentosephosphate pathway proteins and/or the activity of the one or morepentose phosphate pathway proteins is modulated relative to recombinantcells that have not been modified.
 23. The recombinant cells of claim22, wherein the one or more pentose phosphate pathway proteins isselected from the group consisting of transketolase (tktA),transaldolase (talB), ribulose-5-phosphate-epimerase (rpe),ribose-5-phosphate epimerase (rpiA), and phosphofructokinase (pfkA). 24.The recombinant cells of claim 18, wherein the recombinant cells furthercomprise (iv) one or more nucleic acids encoding one or more acetatecycling proteins, wherein the cells have been modified such that theexpression of the one or more nucleic acids encoding the one or moreacetate cycling proteins and/or activity of the one or more acetatecycling proteins is modulated relative to recombinant cells that havenot been modified.
 25. The recombinant cells of claim 24, wherein theone or more acetate cycling proteins is selected from the groupconsisting of acetyl-coenzyme A synthetase (acs), acetate kinase (ackA),acetate transporter/acetate pump (actP) and phosphotransacetylase (pta).26. The recombinant cells of claim 18, wherein the recombinant cellsfurther comprise one or more nucleic acids encoding one or more proteinsselected from the group consisting of: sfcA, maeB, pdhR, aceE, aceF,lpdA, glta, acs, pta, ackA, actP, pfkA, rpe, rpiA, tkta, talB, pgl, edd,and eda, and wherein the cells have been modified such that theexpression of the one or more nucleic acids and/or activity of the oneor more proteins is modulated relative to recombinant cells that havenot been modified.
 27. The recombinant cells of claim 18, wherein therecombinant cells are gram-positive bacterial cells, gram-negativebacterial cells, fungal cells, filamentous fungal cells, algal cells, oryeast cells.
 28. The recombinant cells of claim 27, wherein therecombinant cells are selected from the group consisting of Bacillussubtilis, Streptomyces lividans, Streptomyces coelicolor, Streptomycesgriseus, Escherichia coli, and Pantoea citrea.
 29. The recombinant cellsof claim 27, wherein the recombinant cells are selected from the groupconsisting of Trichoderma reesei, Aspergillus oryzae, Aspergillus niger,Saccharomyces cerevisiae, and Yarrowia lipolytica.
 30. The recombinantcells of claim 18, wherein the isoprenoid is selected from the groupconsisting of a monoterpene, a diterpene, a triterpene, a tetraterpene,a sesquiterpene, and a polyterpene.
 31. The recombinant cells of claim18, wherein the isoprenoid is selected from the group consisting ofabietadiene, amorphadiene, carene, α-famesene, β-farnesene, farnesol,geraniol, geranylgeraniol, linalool, limonene, myrcene, nerolidol,ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpindene, andvalencene.
 32. The recombinant cells of claim 18, wherein the isoprenoidproduction is increased relative to recombinant cells that have not beenmodified in (i)(a) and (i)(b).
 33. The recombinant cells of claim 32,wherein the isoprenoid production is increased by at least 5%, whereinthe increased production of the isoprenoid comprises an increase in: (i)titer, (ii) instantaneous yield, (iii) cumulative yield, (iv) specificproductivity, or (v) cell productivity index.
 34. A method of producingan isoprenoid comprising: (a) culturing the recombinant cell of claim 18under conditions suitable for producing the isoprenoid and (b) producingthe isoprenoid.
 35. The method of claim 34, further comprising (c)recovering the isoprenoid.